Nuclease resistant double-stranded ribonucleic acid

ABSTRACT

This invention relates to modified double-stranded oligoribonucleic acid (dsRNA) having improved stability in cells and biological fluids, and methods of making and identifying dsRNA having improved stability, and of using the dsRNA to inhibit the expression or function of a target gene.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/139,089, filed May 27, 2005; which claims the benefit of U.S.Provisional Application No. 60/574,744, filed May 27, 2004; U.S.Provisional Application No. 60/607,850, filed Sep. 7, 2004; U.S.Provisional Application No. 60/607,790, filed Sep. 8, 2004; and U.S.Provisional Application No. 60/634,860, filed Dec. 9, 2004. The contentof each of these applications is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to modified double-stranded oligoribonucleic acid(dsRNA) having improved stability in cells and biological fluids, andmethods of making and identifying dsRNA having improved stability, andof using the dsRNA to inhibit the expression or function of a targetgene.

BACKGROUND OF THE INVENTION

Many diseases (e.g., cancers, hematopoietic disorders, endocrinedisorders, and immune disorders) arise from the abnormal expression oractivity of a particular gene or group of genes. Similarly, disease canresult through expression of a mutant form of protein, as well as fromexpression of viral genes that have been integrated into the genome oftheir host. The therapeutic benefits of being able to selectivelysilence these abnormal or foreign genes are obvious.

Double-stranded RNA molecules (dsRNA) can block gene expression byvirtue of a highly conserved regulatory mechanism known as RNAinterference (RNAi). Briefly, the RNA III Dicer enzyme processes dsRNAinto small interfering RNA (siRNA) of approximately 22 nucleotides. Onestrand of the siRNA (the “antisense strand”) then serves as a guidesequence to induce cleavage of messenger RNAs (mRNAs) comprising anucleotide sequence which is at least partially complementary to thesequence of the antisense strand by an RNA-induced silencing complexRISC (Hammond, S. M., et al., Nature (2000) 404:293-296). The antisensestrand is not cleaved or otherwise degraded in this process, and theRISC comprising the antisense strand can subsequently effect thecleavage of further mRNAs.

SUMMARY OF THE INVENTION

The present invention discloses double-stranded ribonucleic acid (dsRNA)having improved stability in cells and biological fluids, methods ofmaking and identifying dsRNA having improved stability, as well ascompositions and methods for inhibiting the expression of a target genein a cell using the dsRNA. The present invention also disclosescompositions and methods for treating diseases caused by the expressionor activity of the target gene. The dsRNA of the invention comprises atleast one type of nucleotide modified in the 2′-position, which type ofnucleotide is in a sequence context rendering it particularly prone toendonucleolytic degradation. Such modification renders the dsRNA moreresistant to enzymatic or chemical degradation, and thus more stable andbioavailable than an otherwise identical dsRNA but without the modifiednucleotide.

In one aspect, the invention relates to a double-stranded ribonucleicacid (dsRNA) having increased stability in a biological sample, whereinthe dsRNA comprises at least one of

-   -   (a) 2′-modified uridines in all occurrences of the sequence        motif 5′-uridine-adenine-3′ (5′-ua-3′), or    -   (b) 2′-modified uridines in all occurrences of the sequence        motif 5′-uridine-guanine-3′ (5′-ug-3′), or    -   (c) 2′-modified cytidines in all occurrences of the sequence        motif 5′-cytidine-adenine-3′ (5′-ca-3′), or    -   (d) 2′-modified 5′-most uridines in all occurrences of the        sequence motif 5′-uridine-uridine-3′ (5′-uu-3′), or    -   (e) 2′-modified 5′-most cytidines in all occurrences of the        sequence motif 5′-cytidine-cytidine-3′ (5′-cc-3′), or    -   (f) 2′-modified cytidines in all occurrences of the sequence        motif 5′-cytidine-uridine-3′ (5′-cu-3′), or    -   (g) 2′-modified uridines in all occurrences of the sequence        motif 5′-uridine-cytidine-3′ (5′-uc-3′).

The dsRNA of the invention may comprise at least two, three, four orfive different features of the features (a) through (g) above. Wherethere are at least four different features of the features (a) through(g) above, the four different features may be chosen to be (a), (b),(c), and (d). The dsRNA may comprise at least two or at least threeoccurrences of at least one of the sequence motifs of (a) through (g).The dsRNA may further comprise at least one dinucleotide selected fromthe group of5′-uu-3′,5′-ua-3′,5′-ug-3′,5′-uc-3′,5′-cu-3′,5′-ca-3′,5′-cg-3′,5′-cc-3′,wherein no nucleotide is a 2′-O-modified nucleotide. The 2′-modifiednucleotide can be a 2′-deoxy nucleotide, a 2′-O-methyl nucleotide, a2′-deoxyfluoro nucleotide, a 2′-O-methoxyethyl nucleotide, a 2′-O-NMA, a2′-DMAEOE, a 2′-AP, 2′-hydroxy, 2′-ara-F, or a locked nucleic acidnucleotide, extended nucleic acid, hexose nucleic acid, or cyclohexosenucleic acid. The dsRNA may further comprise a nucleotide overhanghaving 1 to 4 unpaired nucleotides. The overhang may be free ofnucleotides T and U. The overhang may have the nucleotide sequence5′-gcnn-3′, wherein n is a, g, c, u, T, U or nothing. The first pairednucleotide adjacent to the 5′-guanine of the 5′-gcnn-3′ overhang can bea cytidine (c). The unpaired nucleotides may comprise at least onephosphorothioate dinucleotide linkage. The nucleotide overhang can be atthe 3′-end of the antisense RNA strand of the dsRNA. The antisense RNAstrand of the dsRNA can comprise a nucleotide sequence 18-30 nucleotidesin length which is complementary to the sense strand. The antisense andsense RNA strands of the dsRNA can be connected by a chemical linker.

In a second aspect, the invention relates to a method of preparing apharmaceutical composition, comprising formulating the dsRNA of theinvention in a pharmaceutically acceptable carrier.

In a third aspect, the invention relates to a pharmaceutical compositionfor inhibiting the expression of a target gene in a mammal, comprisingat least one dsRNA of the invention and a pharmaceutically acceptablecarrier. The pharmaceutically acceptable carrier may be an aqueoussolution, e.g. physiological saline.

In a fourth aspect, the invention relates to a method for inhibiting theexpression of a target gene in a cell, comprising the steps ofintroducing a dsRNA of the invention into the cell, and maintaining thecell for a time sufficient to obtain inhibition of expression of thetarget gene in the cell, wherein methods of treatment or diagnosis to beperformed on a human or animal body are excluded. The cell may be amammalian cell.

In a fifth aspect, the invention relates to a method for making adouble-stranded RNA (dsRNA) with high stability in biological samplesfor inhibiting the expression of a target gene comprising the steps of:

-   -   (a) selecting one or more nucleotide sequences of between 18 and        30 nucleotides in length from the nucleotide sequence of the        mRNA resulting from the transcription of the target gene; and    -   (b) synthesizing one or more dsRNAs, wherein one strand        comprises a sequence complementary to one of the nucleotide        sequences selected in a.; and    -   (c) testing said one or more dsRNAs for their capability to        inhibit the expression of the target gene in a biological        sample; and    -   (d) selecting one of the one or more dsRNAs of c. possessing the        capability to inhibit the expression of the target gene in a        biological sample; and    -   (e) in the dsRNA selected in d., identifying in the nucleotide        sequences of the sense strand as well as the antisense strand        all occurrences of the dinucleotides 5′-ua-3′,5′-ca-3′,5′-ug-3′,        5′-uu-3′,5′-cc-3′,5′-uc-3′ and 5′-cu-3; and    -   (f) synthesizing a dsRNA, wherein the 5′-uridines and/or        5′-cytidines in all occurrences of at least one of the        dinucleotides identified in e. is replaced by a 2′-modified        uridine and/or cytidine, respectively.

In one embodiment of this method, the 5′-uridines and/or 5′-cytidines inall occurrences of at least two of the dinucleotides identified in e.are replaced by a 2′-modified uridine and/or cytidine, respectively.Preferably, the 5′-uridines and/or 5′-cytidines in all occurrences of atleast three of the dinucleotides identified in e. are replaced by a2′-modified uridine and/or cytidine, respectively. More preferably, the5′-uridines and/or 5′-cytidines in all occurrences of at least four ofthe dinucleotides identified in e. are replaced by a 2′-modified uridineand/or cytidine, respectively. Said four dinucleotides may, for example,be 5′-ua-3′,5′-ug-3′,5′-uu-3′, and 5′-ca-3′. Most preferably, the5′-uridines and/or 5′-cytidines in all occurrences of at least five ofthe dinucleotides identified in e. are replaced by a 2′-modified uridineand/or cytidine, respectively.

In a sixth aspect, the invention relates to a method of treating adisease caused by expression of a target gene in a subject, said methodcomprising administering to said subject a pharmaceutical compositioncomprising the dsRNA of the invention and a pharmaceutically acceptablecarrier. Said subject may, for example, be a human.

In a seventh aspect, the invention relates to a method of increasing thenuclease resistance of a dsRNA, comprising the steps of:

-   -   (a) identifying in the nucleotide sequences of the sense strand        as well as the antisense strand of the dsRNA all occurrences of        the dinucleotides        5′-ua-3′,5′-ca-3′,5′-ug-3′,5′-uu-3′,5′-cc-3′,5′-uc-3′ and        5′-cu-3; and    -   (b) replacing the 5′-uridines and/or 5′-cytidines in all        occurrences of at least one of the dinucleotides identified        in (a) by 2′-modified uridines and/or cytidines, respectively.

In a preferred embodiment of said method, all of the 5′-uridines and/or5′-cytidines in at least two of the dinucleotides identified in (a) arereplaced by 2′-modified uridines and/or cytidines, respectively.Preferably, all of the 5′-uridines and/or 5′-cytidines in at least threeof the dinucleotides identified in (a) are replaced by 2′-modifieduridines and/or cytidines, respectively. More preferably, all of the5′-uridines and/or 5′-cytidines in at least four of the dinucleotidesidentified in (a) are replaced by 2′-modified uridines and/or cytidines,respectively. Said four dinucleotides may, for example, be5′-ua-3′,5′-ug-3′,5′-uu-3′, and 5′-ca-3′. Most preferably, all of the5′-uridines and/or 5′-cytidines in at least five of the dinucleotidesidentified in (a) are replaced by 2′-modified uridines and/or cytidines,respectively.

In another embodiment, the replacement of uridines and cytidines by2′-modified uridines and cytidines, respectively, is carried outstepwise, wherein

-   -   (a) in one step, all uridines in a 5′-ua-3′ sequence context are        replaced by 2′-modified uridines, and,    -   (b) in an optional further step, all uridines in a 5′-ua-3′        sequence context and all cytidines in a 5′-ca-3′ sequence        context are replaced by the respective 2′-modified nucleotides,        and,    -   (c) in an optional further step, all uridines in a 5′-ua-3′        sequence context and all cytidines in a 5′-ca-3′ sequence        context and all uridines in a 5′-ug-3′ are replaced by the        respective 2′-modified nucleotides, and,    -   (d) in an optional further step, all uridines in a 5′-ua-3′        sequence context and all cytidines in a 5′-ca-3′ sequence        context and all uridines in a 5′-ug-3′ and all uridines in a        5′-uu-3′ sequence context are replaced by the respective        2′-modified nucleotides, and,    -   (e) in an optional further step, all uridines in a 5′-ua-3′        sequence context and all cytidines in a 5′-ca-3′ sequence        context and all uridines in a 5′-ug-3′ and all uridines in a        5′-uu-3′ sequence context and all cytidines in a 5′-cc-3′        sequence context are replaced by the respective 2′-modified        nucleotides, and,    -   (f) in an optional further step, all uridines in a 5′-ua-3′        sequence context and all cytidines in a 5′-ca-3′ sequence        context and all uridines in a 5′-ug-3′ and all uridines in a        5′-uu-3′ sequence context and all cytidines in a 5′-cc-3′        sequence context and all uridines in a 5′-uc-3′ sequence context        are replaced by the respective 2′-modified nucleotides, and,    -   (g) in an optional further step, all uridines in a 5′-ua-3′        sequence context and all cytidines in a 5′-ca-3′ sequence        context and all uridines in a 5′-ug-3′ and all uridines in a        5′-uu-3′ sequence context and all cytidines in a 5′-cc-3′        sequence context and uridines in a 5′-uc-3′ sequence context and        all cytodines in a 5′-cu-3′ are replaced by the respective        2′-modified nucleotides,

wherein at least one of steps (a) through (g) is actually performed andwherein, after each step, the stability of the modified dsRNA(s) inbiological samples is determined.

In an eighth aspect, the invention relates to a method to identify adsRNA with increased stability in biological samples, comprising thesteps of:

-   -   (a) synthesizing a first dsRNA of claim 1 and a second dsRNA        identical to the first dsRNA except that it does not comprise        the 2′-modified nucleotides of the dsRNA of claim 1, and    -   (b) determining the stability of said first and said second        dsRNA in a biological sample by contacting both under identical        conditions with the biological sample, and monitoring their        degradation,

whereby, where the first dsRNA is degraded less rapidly than the seconddsRNA, a dsRNA with increased stability in biological samples isidentified.

In a ninth aspect, the invention relates to a method of treating adisease caused by expression of a target gene in a subject, said methodcomprising administering to said subject a pharmaceutical compositioncomprising the dsRNA of claim of the first aspect of the invention, anda pharmaceutically acceptable carrier.

While RNA interference using dsRNA has been shown to be an effectivemeans for selective gene silencing, RNA can have less than desiredstability in some bodily fluids, particularly in serum. Thus, RNA,including dsRNA, can be degraded between the time it is administered toa subject and the time it enters a target cell. Even within the cell,RNA can undergo rapid degradation by nucleases. Methods of the inventioncan provide more stable or nuclease resistant dsRNAs that can offerbetter bioavailability and hence improved effectiveness. This is animprovement over the current insufficient methods for stabilizing dsRNAagainst degradation.

Thus despite significant recent developments in the field of RNAinterference, there remains a need for a more effective dsRNA moleculethat can selectively and efficiently silence a target gene. Morespecifically, a dsRNA molecule having enhanced resistance to chemicaland/or enzymatic degradation, and hence improved stability in biologicalsamples and bioavailability, and which can be readily andcost-effectively synthesized would be highly desirable. Compositionscomprising such agents would be useful for treating diseases caused byabnormal expression or activity of a gene.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a gel electrophoretic separation, stained using the “stainsall” reagent, of the dsRNA referred to herein as AL-DP-5048 untreated(unb) or treated by incubation with human serum for 0, 1, 3, or 6 hours.AL-DP-5048 is an all-ribonucleic acid bearing no 2′-modifications orphosphorothioates.

FIG. 2 shows a gel electrophoretic separation, stained using the “stainsall” reagent, of the dsRNA referred to herein as AL-DP-5437 untreated(unb) or treated by incubation with human serum for 0, 1, 3, or 6 hours.AL-DP-5437 is identical in nucleotide sequence to AL-DP-5048, exceptthat is bears phosphorothioate linkages between positions 20 an 21 ofthe sense strand, and between positions 21 and 22, as well as between 22and 23, of the antisense strand, protecting this dsRNA againstexonucleolytic attack.

FIG. 3 shows a gel electrophoretic separation, stained using the “stainsall” reagent, of the dsRNA referred to herein as AL-DP-5392 untreated(unb) or treated by incubation with human serum for 0, 1, 3, or 6 hours.AL-DP-5392 is identical in nucleotide sequence to AL-DP-5437, exceptthat is bears additional 2′-modifications in the 5′-most uridines orcytidines of all occurrences of the sequence motifs5′-ua-3′,5′-ug-3′,5′-uu-3′, and 5′-ca-3′, protecting this dsRNA againstexonucleolytic and endonucleolytic attack.

FIG. 4 shows the synthesis and structure of RNA strands conjugated to acholesteryl moiety as described herein.

FIG. 5 shows a gel electrophoretic separation, stained using the “stainsall” reagent, of an unmodified (control) dsRNA (referred to as“GE1s/GE1as”) after 0, 15, 30, 60, 120 and 240 minutes of incubation inserum.

FIG. 6 shows a gel electrophoretic separation, stained using the “stainsall” reagent, of a nuclease resistant dsRNA of the present invention(referred to as “GE7s/GE7 as”) after 0, 15, 30, 60, 120 and 240 minutesof incubation in serum.

FIG. 7 shows a gel electrophoretic separation, stained using the “stainsall” reagent, of a nuclease resistant dsRNA of the present invention(referred to as “GE7s/GE8 as”) after 0, 15, 30, 60, 120 and 240 minutesof incubation in serum.

FIG. 8 shows a gel electrophoretic separation, stained using the “stainsall” reagent, of a nuclease resistant dsRNA of the present invention(referred to as “GE7s/GE9 as”) after 0, 15, 30, 60, 120 and 240 minutesof incubation in serum.

FIG. 9 shows a gel electrophoretic separation, stained using the “stainsall” reagent, of a nuclease resistant dsRNA of the present invention(referred to as “GE7s/GE7 as”) after 0, 15, and 30 minutes, and 1, 2, 4,8, 16 and 24 hours of incubation in serum.

FIG. 10 shows a gel electrophoretic separation, stained using the“stains all” reagent, of a nuclease resistant dsRNA of the presentinvention (referred to as “GE7s/GE8 as”) after 0, 15, and 30 minutes,and 1, 2, 4, 8, 16 and 24 hours of incubation in serum.

FIG. 11 shows a gel electrophoretic separation, stained using the“stains all” reagent, of a nuclease resistant dsRNA of the presentinvention (referred to as “GE7s/GE9 as”) after 0, 15, and 30 minutes,and 1, 2, 4, 8, 16 and 24 hours of incubation in serum.

FIG. 12 shows a gel electrophoretic separation, stained using the“stains all” reagent, of a nuclease resistant dsRNA of the presentinvention (referred to as “GE7s/GE10as”) after 0, 15, 30, 60, 120 and240 minutes of incubation in serum. The sequence of one major fragmentderived from endonucleolytic attack on the antisense strand(3′-CgcuAGUGGGAGGAAUUuAuAAA-5′, SEQ ID NO: 8) between positions 8 and 9(5′-U⁸|A⁹-3′), as determined by mass spectrometry, is shown.

FIG. 13 shows a gel electrophoretic separation, stained using the“stains all” reagent, of a nuclease resistant dsRNA of the presentinvention (referred to as “GE7s/GE11 as”) after 0, 15, 30, 60, 120 and240 minutes of incubation in serum.

FIG. 14 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNA referred to herein as “GE1s/GE7 as”after 0, 15, 30, 60, 120 and 240 minutes of incubation in serum.

FIG. 15 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNA referred to herein as “GE1s/GE8 as”after 0, 15, 30, 60, 120 and 240 minutes of incubation in serum.

FIG. 16 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNA referred to herein as “GE1s/GE9 as”after 0, 15, 30, 60, 120 and 240 minutes of incubation in serum.

FIG. 17 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNA referred to herein as “GE1s/GE10as”after 0, 15, 30, 60, 120 and 240 minutes of incubation in serum.

FIG. 18 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNA referred to herein as “GE1s/GE11 as”after 0, 15, 30, 60, 120 and 240 minutes of incubation in serum.

FIG. 19 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNA referred to herein as “GE7s/GE1as”after 0, 15, 30, 60, 120 and 240 minutes of incubation in serum.

FIG. 20 shows schematically the difference between the products of exo-and endonucleolytic degradation of RNA. Exonucleolytic degradationrequires a phosphodiester bond between the last and penultimate bases onthe 3′-end of the RNA strand and may be hindered by substituting thephosphodiester group by a phosphorothioate. Endonucleolytic degradationproceeds via nucleophilic attack of a 2′-hydroxyl group on its3′-phosphorous, and may be hindered by substituting the 2′-hydroxylgroup by, e.g. a 2′-O-methyl group.

FIG. 21 shows schematically the process by which the products ofnucleolytic degradation of dsRNA in serum were identified using liquidchromatography/mass spectrometry (LC/MS). DsRNA was incubated with mouseor human serum for various time spans, total RNA was isolated from theincubation mix and fragments separated on an LC under denaturingconditions. The LC output was fed into a mass spectrometer and the massof ions appearing in mass spectrometric analysis was compared to thepredicted masses of all possible fragments for a given dsRNA.

FIG. 22 shows in more detail the LC/MS analysis of fragments of dsRNAafter incubation with serum. The upper panel depicts a plot of the totalion current measured by the mass spectrometer as a function of time; thepeaks corresponding to the various RNA fragments of different lengthsare clearly separated. The center panel depicts the result of a massseparation of ions generated from an RNA fragment appearing as one peakin the upper panel, plotting relative abundancy of ionic species vs. themass to charge ratio of each ionic species. The lower panel shows theresult of deconvoluting the data shown in the middle panel, i.e.correcting mass to charge for charge. The two main peaks at m/z=1284.8and 1713.4 thereby collapse into a single peak corresponding to a massof the parent RNA [M-H]=5142.8 DA. This observed mass corresponds withinerror limits to the predicted mass of the fragment5′-ucgaaguacucagcgu-3′ (SEQ ID NO:1), which may be derived from theantisense strand of the dsRNA (SEQ ID NO:185, SEQ ID NO:184), referredto herein as LC1s/LC1as by endonucleolytic cleavage of the 5′-ua-3′dinucleotide at positions 16-17.

FIG. 23 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5024,AL-DP-5388, and AL-DP-5448 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. These dsRNAs are identical innucleotide sequence, except that AL-DP-5024 is an unmodifiedribonucleotide, AL-DP-5388 bears 2′-O-methyl-modifications of the5′-uridine or 5′-cytidine in all occurrences of the sequence motifs5′-ua-3′,5′-ug-3′,5′-ca-3′, and 5′-uu-3′, as well as in positions 22 and23 of the antisense strand, phosphorothioate linkages between positions20 an 21 of the sense strand, and between positions 22 and 23 of theantisense strand (labeled “2′-OMe”), and AL-DP-5448 bears2′-deoxy-2′-fluoro-modifications of the 5′-uridine or 5′-cytidine in alloccurrences of the sequence motifs 5′-ua-3′,5′-ug-3′, 5′-ca-3′, and5′-uu-3′,2′-O-methyl-modifications in positions 22 and 23 of theantisense strand unless already 2′-deoxy-2′-fluoro-modified,phosphorothioate linkages between positions 20 an 21 of the sensestrand, and between positions 22 and 23 of the antisense strand (labeled“2′-F”).

FIG. 24 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5013,AL-DP-5387, and AL-DP-5447 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

FIG. 25 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5084,AL-DP-5394, and AL-DP-5454, untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

FIG. 26 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5094,AL-DP-5397, and AL-DP-5457 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

FIG. 27 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5097,AL-DP-5398, and AL-DP-5458 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

FIG. 28 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5093,AL-DP-5396, and AL-DP-5456 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

FIG. 29 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5098,AL-DP-5399, and AL-DP-5459 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

FIG. 30 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5089,AL-DP-5395, and AL-DP-5455 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

FIG. 31 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5030,AL-DP-5389, and AL-DP-5449 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

FIG. 32 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5035,AL-DP-5390, and AL-DP-5450 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

FIG. 33 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5046,AL-DP-5391, and AL-DP-5451 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

FIG. 34 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5048,AL-DP-5392, and AL-DP-5452 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

FIG. 35 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5002,AL-DP-5386, and AL-DP-5446 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

FIG. 36 shows a gel electrophoretic separation, stained using the“stains all” reagent, of the dsRNAs referred to herein as AL-DP-5049,AL-DP-5393, and AL-DP-5453 untreated (N.s.) or treated by incubationwith human serum for 0, 1, 3, or 6 hours. Labels “2′-OMe” and “2′-F”have the meaning as described for FIG. 23.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses modified double-stranded ribonucleicacid (dsRNA) having improved stability in cells and biological fluidscompared to unmodified dsRNA recognizing the same target RNA, methods ofmaking the modified dsRNA, as well as compositions and methods forinhibiting the expression of a target gene in a cell or mammal using thedsRNA. The present invention also discloses compositions and methods fortreating diseases in organisms caused by expression of a target geneusing the modified dsRNA.

dsRNA directs the sequence-specific degradation of mRNA through aprocess known as RNA interference (RNAi). The process occurs in a widevariety of organisms, including mammals and other vertebrates. The dsRNAof the invention comprises an antisense strand and a sense strand,wherein the antisense strand has a nucleotide sequence which iscomplementary to a target RNA, and comprises at least one of:2′-modified uridines in all occurrences of the sequence motif5′-uridine-adenine-3′ (5′-ua-3′), or 2′-modified uridines in alloccurrences of the sequence motif 5′-uridine-guanine-3′ (5′-ug-3′), or2′-modified cytidines in all occurrences of the sequence motif5′-cytidine-adenine-3′ (5′-ca-3′), or 2′-modified 5′-most uridines inall occurrences of the sequence motif 5′-uridine-uridine-3′ (5′-uu-3′),or 2′-modified 5′-most cytidines in all occurrences of the sequencemotif 5′-cytidine-cytidine-3′ (5′-cc-3′), or 2′-modified cytidines inall occurrences of the sequence motif 5′-cytidine-uridine-3′ (5′-cu-3′),or 2′-modified uridines in all occurrences of the sequence motif5′-uridine-cytidine-3′ (5′-uc-3′). The present inventors have discoveredthat dsRNAs comprising such modifications (i.e., a modified dsRNA) havesignificantly improved stability in biological samples compared to theirunmodified dsRNA counterparts. The dinucleotide(s) chemically modifiedin the 2′-position may be strategically placed within the dsRNA foroptimal stability and without affecting the interference activity. Thepresent invention encompasses these modified dsRNAs, compositionscomprising the modified dsRNAs, and the use of these compositions forspecifically inactivating gene function. The use of modified dsRNAshaving improved resistance to enzymatic degradation (i.e., increased invivo half-life), and hence improved bioavailability, facilitates thetargeted degradation of mRNA of genes that are implicated in a widevariety of disease processes. Because of the improved bioavailability ofthe modified dsRNAs, less dsRNA is required to produce the desired RNAinterference effect. Thus, the methods and compositions of the presentinvention comprising the modified dsRNAs are useful for treatingdiseases and disorders caused by the expression or activity of aparticular gene.

The invention also relates to methods of making and using the dsRNAs andcompositions containing dsRNAs having improved stability in biologicalsamples to inhibit the expression of a target gene, as well ascompositions and methods for treating diseases and disorders caused bythe expression of the target gene. Exemplary pharmaceutical compositionscomprise a modified dsRNA having an antisense nucleotide sequence of,for example, 18 to 25 nucleotides in length, preferably 22 nucleotidesin length, and which is substantially complementary to a region of anmRNA transcript of the target gene, together with a pharmaceuticallyacceptable carrier.

Accordingly, certain aspects of the present invention relate topharmaceutical compositions comprising the modified dsRNA of the presentinvention together with a pharmaceutically acceptable carrier, methodsof making and using the modified dsRNA to inhibit expression of a targetgene, and methods of making and using the modified dsRNA to treatdiseases caused by the expression or activity of a particular gene.Methods of identifying dsRNA having improved stability are alsodisclosed.

1. Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below.

As used herein, “target gene” refers to a section of a DNA strand of adouble-stranded DNA that is complementary to a section of a DNA strand,including all transcribed regions, that serves as a template fortranscription. A target gene, usually the sense strand, is a gene whoseexpression is to be selectively inhibited or silenced through RNAinterference. The term “target gene” specifically encompasses anycellular gene or gene fragment whose expression or activity isassociated with a disease or disorder (e.g., an oncogene), as well asany foreign or exogenous gene or gene fragment whose expression oractivity is associated with a disease, such as a gene from a pathogenicorganism (e.g., a viral or pro-viral gene, viroid, or plasmodium). Thetarget gene may be a viral gene, for example a (+) strand RNA virus suchas a Hepatitis C Virus (HCV).

As used herein, the term “double-stranded RNA” or “dsRNA” (also called“siRNA” or “iRNA agent”) refers to a ribonucleic acid molecule having aduplex structure comprising two at least partly, and preferably fully,mutually complementary and anti-parallel nucleic acid strands. Thestrands forming the duplex structure may be comprised on separatemolecules, or they may be part of a single molecule, i.e. linked by oneor more covalent bonds. Not all nucleotides of a dsRNA must exhibitWatson-Crick base pairs; the two RNA strands may be substantiallycomplementary (i.e., having no more than two, preferably no more thanone, nucleotide mismatches per 10 contiguous nucleotide base pairs). TheRNA strands may have the same or a different number of nucleotides.Preferably, the complementary region between the antisense and sense RNAstrands is at least 10, 15, 18, 19, 20, 22 or 23 nucleotides, but nomore than 23, 24, 25, 28, 30, 35, or 49 nucleotides, in length. The term“duplex” or “duplex structure” refers to the region of the dsRNAmolecule wherein the two separate RNA strands or the singleself-complementary RNA strand are complementary, and thus hybridize toform a double-stranded structure.

The term “antisense RNA strand” (also referred to herein as the“antisense strand”) refers to the strand of a dsRNA that iscomplementary to an mRNA transcript that is formed during expression ofthe target gene, or its processing products, or a region (such as the3′-UTR) of a (+) strand RNA virus. As used herein, the term “antisensenucleotide sequence” refers to the region on the antisense RNA strandthat is complementary to a region of an mRNA transcript of the targetgene or a region (e.g., 3′-UTR) of a (+) strand RNA virus.

As used herein, “modified dsRNA” refers to a dsRNA molecule thatcomprises at least one alteration. The modified dsRNAs of the presentinvention include at least one of: 2′-modified uridines in alloccurrences of the sequence motif 5′-uridine-adenine-3′ (5′-ua-3′), or2′-modified uridines in all occurrences of the sequence motif5′-uridine-guanine-3′ (5′-ug-3′), or 2′-modified cytidines in alloccurrences of the sequence motif 5′-cytidine-adenine-3′ (5′-ca-3′), or2′-modified 5′-most uridines in all occurrences of the sequence motif5′-uridine-uridine-3′ (5′-uu-3′), or 2′-modified 5′-most cytidines inall occurrences of the sequence motif 5′-cytidine-cytidine-3′(5′-cc-3′), or 2′-modified cytidines in all occurrences of the sequencemotif 5′-cytidine-uridine-3′ (5′-cu-3′), or 2′-modified uridines in alloccurrences of the sequence motif 5′-uridine-cytidine-3′ (5′-uc-3′).

The terms “modified nucleotide,” “substituted nucleotide,” and“additional nucleotide,” as used herein, refer to a nucleotide which hasbeen altered, or added to a nucleic acid sequence of a dsRNA inreplacement of another nucleotide, or simply added to such nucleic acidsequence, respectively, to render the dsRNA more resistant to nucleases(i.e., more stable) than a naturally occurring dsRNA or a chemicallysynthesized dsRNA that recognizes the same target sequence but lacks thealteration, substitution, or addition of such nucleotide. Exemplarymodifications that generate modified, substituted, or additionalnucleotides that increase the stability of the dsRNA include, forexample, replacing a naturally occurring or wild-type nucleotide with a2′-modified nucleotide, as defined below (e.g., replacing a uridine witha 2′-O-methyl uridine or a 2′-deoxy-2′-fluoro uridine), addition of oneor more unpaired nucleotides or dinucleotides at an end of the doublestranded structure (e.g., incorporating a 5′-GC-3′ sequence which doesnot normally flank the target RNA sequence), chemical modification of anucleotide (e.g., replacing a 2′-hydroxyl group on the sugar moiety witha 2′-O-methyl group or a 2′-deoxy-2′-fluoro group), and alteration of anucleotide linkage (e.g., replacing a phosphodiester bond with aphosphorothioate bond). The “modified dsRNA” of the present inventioncontains a modification in its chemical structure that produces ameasurable increase in stability as compared to an identical dsRNAwithout the substituted or modified nucleotide.

Particularly, the term “2′-modified nucleotide,” as used herein, refersto a nucleotide which has been modified as to prevent the enzymaticcleavage of the phosphate backbone of an RNA at or near the nucleotideso modified. Often, such cleavage is mediated by the 2′-OH of anucleotide via formation of a cyclic phosphate. Removing or replacingthe 2′-OH group can effectively block cleavage, e.g. by not allowing theformation of the cyclic phosphate. Modifications within the scope of theinstant invention include, without limitation, the removal of the 2′-OH,resulting in a 2′-deoxy nucleotide, or the replacement of the 2′-OH byOR¹, O(CH₂CH₂O)_(m)CH₂CH₂OR¹; O(CH₂)_(n)R²; O(CH₂)_(n)OR², halogen; NH₂;NHR¹; N(R¹)₂; NH(CH₂CH₂NH)_(m)CH₂CH₂NHR⁹; NHC(O)R¹; cyano; mercapto,SR¹; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl,alkenyl, or alkynyl, each of which may be optionally substituted withhalogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl,alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino,acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl,carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido,arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, aryloxy, cyano, orureido; R¹ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,heteroaryl, amino acid, or sugar; R² is NH₂, alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid; m is 0-1,000,000, and n is 0-20; or the 2′-oxygenmay be used to form an [—O—CH₂—] covalently bound bridge between thesugar 2′ and 4′ carbon atoms (commonly, a “locked nucleic acid”). Theskilled person will be aware of further modifications to achieve thedesired effect of blocking endonuclease cleavage. Furthermore,additional such modifications developed in the future but are equivalentin effect are within the scope of the present invention.

As used herein, the terms “stability” and “stable” refer to resistanceto degradation, e.g., by chemicals or nucleases, specificallyendonucleases, which normally degrade RNA molecules. The modified dsRNAmolecules of the present invention have a measurable change instability, and hence longer half-lives, than their unmodified dsRNAcounterparts.

As used herein, a “measurable change” or “measurable increase” in dsRNAstability refers to a quantity that is empirically determined and thatwill vary depending upon the method used to monitor dsRNA stability. Thepresent invention encompasses any difference between the test andcontrol combinations in any measurable physical parameter (e.g.,stability in serum), where the difference is greater than expected dueto random statistical variation. For example, a modified dsRNA of thepresent invention has “increased stability” or is “more stable” than acontrol dsRNA when the amount of non-degraded modified dsRNA is at least10%, 25%, 35%, or 50% more than that of a control dsRNA after incubationof both dsRNAs with, for example, a biological sample.

As used herein, the term “control” refers to a dsRNA molecule that isstructurally similar or identical to a modified dsRNA molecule exceptthat it lacks at least one of the modified, substituted, or addednucleotides present in the modified dsRNA. As will be appreciated bythose skilled in the art, the “control” dsRNA will depend upon theexperimental design, and will vary depending upon the structural featureor features being evaluated. Preferably, the modified dsRNAs of theinvention are substantially resistant to enzymatic (e.g., endonucleaseand exonuclease) degradation, more preferably substantially resistant toendonucleases, and most preferably highly resistant to endonucleases.Also as used herein, the term “control cell” refers to a cell that iscapable of expressing a target gene, and which does not include themodified dsRNA.

As used herein, a modified dsRNA is “substantially resistant” tonucleases when it is at least about 2-fold more resistant to attack byone nuclease, or a set of nucleases, and is “highly nuclease resistant”when it is at least about 5-fold more resistant to a nuclease or a setof nucleases than a control dsRNA.

As used herein, the term “biological sample” refers to a whole organismor a subset of its tissues, cells or component parts (e.g., body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). “A biological sample” further refers toa homogenate, lysate or extract prepared from a whole organism or asubset of its tissues, cells or component parts, or a fraction orportion thereof, including but not limited to, for example, plasma,serum, spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. Most often, the sample has been removedfrom a multi-cellular organism, such as an animal, but the term“biological sample” can also refer to cells or tissue analyzed in vivo,i.e., without removal from the multi-cellular organism, or to cellspropagated and/or analyzed in vitro, which may or may not stem from amulti-cellular organism, e.g., bacterial cells. Typically, a “biologicalsample” will contain cells from the animal, but the term can also referto non-cellular biological material, such as non-cellular fractions ofblood, saliva, or urine, that can be used to measure adisease-associated polynucleotide or polypeptide levels. “A biologicalsample” further refers to a medium, such as a nutrient broth or gel inwhich an organism has been propagated, which contains cellularcomponents, such as proteins or nucleic acid molecules.

As used herein, the term “subject” refers to an organism to which thenucleic acid molecules of the invention can be administered. In oneembodiment, a subject is a mammal (such as a mouse, rat, human, ornonhuman primate) or mammalian cells. In another embodiment, a subjectis a human or human cells.

“Introducing into” means facilitating uptake or absorption into a cell,as is understood by those skilled in the art. Absorption or uptake ofdsRNA can occur through unaided diffusive or cellular processes, or bymeans of auxiliary agents or devices, such as viral and syntheticvectors. For example, for introduction of dsRNA into cells in vivo,dsRNA can be injected into a tissue site or administered systemically.Introduction of dsRNA into cells in vitro includes methods known in theart such as electroporation and lipofection.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure when a3′-end of one RNA strand extends beyond the 5′-end of the othercomplementary strand, or vice versa. “3′-blunt”- or “5′-blunt” or “bluntend” means that there are no unpaired nucleotides at that end of thedsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNAthat is double stranded over its entire length, i.e., no nucleotideoverhang at either end of the molecule.

The term “terminal base pair,” as used herein, means the last nucleotidebase pair on one end of the duplex region of the dsRNA. Thus, where thedsRNA is blunt ended (i.e., has no nucleotide overhangs), the lastnucleotide base pairs at both ends of the dsRNA are terminal base pairs.Where a dsRNA has a nucleotide overhang at one or both ends of theduplex structure, the last nucleotide base pair(s) immediately adjacentthe nucleotide overhang(s) is (are) the terminal base pair(s) at thatend(s) of the dsRNA.

The term “position” with respect to a nucleotide forming part of anoligonucleotide refers to the relative position of the nucleotide withinthe oligonucleotide, counting all nucleotides in integers from the5′-end of the oligonucleotide, position 1 being the 5′-terminalnucleotide. For example, the oligonucleotide 5′-aucg-3′ comprises anadenosine monophosphate in position 1, uridine monophosphate in position2, cytidine monophosphate in position 3, and guanosine monophosphate inposition 4.

The term “sequence motif” or “sequence context”, as used herein, refersto a certain nucleotide sequence of at least 2 nucleotides comprised ina larger oligonucleotide sequence. A sequence motif may occur once in anoligonucleotide sequence, or it may occur any number of times. Forexample, the oligonucleotide 5′-aucaucaug-3′ comprises three occurrencesof the sequence motif 5′-au-3′, two occurrences of the sequence motifs5′-uc-3′ and 5′-ca-3′, and one occurrence of the sequence motif5′-ug-3′.

2. Double-Stranded Ribonucleic Acids (dsRNA) with Improved Stability inBiological Samples

In one embodiment, the invention relates to a double-strandedribonucleic acid (dsRNA) having improved resistance to chemical and/ornuclease digestion, and thus increased stability in biological samplesand a longer in vivo half-life. Increasing the in vivo half-life of thedsRNA results in enhanced bioavailability, and hence improvedeffectiveness in inhibiting expression or activity of a target gene.Thus, the present invention is based, at least in part, on improving theefficiency of dsRNA as a therapeutic agent, by increasing the stabilityin biological samples of the dsRNA, while maintaining the ability of thedsRNA to mediate RNA interference in vivo.

The dsRNA of the present invention comprises at least one substituted ormodified nucleotide that enhances the stability in biological samples ofthe dsRNA compared to an identical dsRNA that recognizes the same targetsequence but that lacks the substituted or modified nucleotide. Asdiscussed below, the substituted or modified nucleotide is strategicallylocated within the dsRNA for optimal stability, yet having little or noeffect on interference activity. The dsRNA of the present invention mayfurther comprise at least one phosphorothioate internucleoside linkagethat preferably enhances the stability in biological samples of thedsRNA. As discussed in detail below, the dsRNA can be synthesized bystandard methods known in the art, e.g., using an automated DNAsynthesizer, such as those commercially available from Biosearch,Applied Biosystems, Inc., or other manufacturers known to the skilledperson.

The present invention is based, in part, on the discovery that dsRNAmolecules modified to contain at least one of 2′-modified uridines inall occurrence s of the sequence motif 5′-uridine-adenine-3′ (5′-ua-3′),or 2′-modified uridines in all occurrences of the sequence motif5′-uridine-guanine-3′ (5′-ug-3′), or 2′-modified cytidines in alloccurrences of the sequence motif 5′-cytidine-adenine-3′ (5′-ca-3′), or2′-modified 5′-most uridines in all occurrences of the sequence motif5′-uridine-uridine-3′ (5′-uu-3′), or 2′-modified 5′-most cytidines inall occurrences of the sequence motif 5′-cytidine-cytidine-3′(5′-cc-3′), or 2′-modified cytidines in all occurrences of the sequencemotif 5′-cytidine-uridine-3′ (5′-cu-3′), or 2′-modified uridines in alloccurrences of the sequence motif 5′-uridine-cytidine-3′ (5′-uc-3′),have significantly enhanced stability in biological samples. Thus, forexample, all uridines in the sequence context 5′-uridine-adenine-3′(5′-ua-3′) can be replaced with 2′-O-methyl uridines to increase thestability of a dsRNA as compared to the stability of a correspondingdsRNA comprising unmethylated uridine (2′-OH). Furthermore, theinventors have found it to be particularly advantageous to replace alluridines and cytidines in sequence contexts 5′-ua-3′,5′-ug-3′,5′-uu-3′,and 5′-ca-3′. The present inventors have discovered that incorporationof a, for example, 2′-O-methyl modification into a dsRNA comprisingcertain sequence motifs particularly prone to enzymatic cleavagesignificantly enhances the stability as compared to the correspondingunmodified dsRNA.

Cleavage of RNA or dsRNA by nucleolytic enzymes requires the formationof an enzyme-substrate complex, i.e., a particularnuclease-oligonucleotide complex. The nucleases generally requirespecific binding sites on the oligonucleotide for appropriateattachment. If the binding sites are removed or blocked, such that thenucleases are unable to attach to the oligonucleotide, theoligonucleotide will become nuclease resistant. This concept is wellestablished particularly for the protection of oligonucleotides fromdegradation of exonucleases, enzymes that degrade oligonucleotidesexclusively from their ends.

The skilled person is well aware that incorporation of, for example,2′-modified nucleotides, e.g. 2′-O-methylated nucleotides, at, orphosphorothioate linkages between, the two or three 3′-most and/or5′-most, but particularly 3′-most, nucleotide positions of anoligonucleotide has been shown to be an efficient means for protectionagainst exonucleolytic degradation. However, the present inventors havefound that protection against exonucleolytic degradation is notsufficient to confer to a dsRNA the desired stability in biologicalsamples. A dsRNA that is protected from exonucleolytic attack is stilldegraded quickly in biological samples by the action of omnipresentendonucleolytic enzymes. For example, FIG. 1, FIG. 2, and FIG. 3 show,respectively, the incubation of one unmodified dsRNA, a dsRNA protectedagainst exonucleolytic attack by incorporation of phosphorothioatelinkages between the 3′-terminal nucleotides, and a dsRNA that bears, inaddition to phosphorothioate linkages between the 3′-terminalnucleotides, 2′-O-mthyl modifications in all occurrences of certainsequence motifs found particularly prone to endonucleolytic attack,namely 5′-ua-3′,5′-ug-3′,5′-uu-3′, and 5′-ca-3′. The figures aptlydemonstrate that only the latter survives to any measurable amountbeyond 1 hour incubation in human serum.

The incorporation of 2′-modified non-terminal nucleotides has beenreported to increase the stability of the modified dsRNA by increasingits resistance to nucleases, see Chiu, Y. L., and Rana, T. M., RNA(2003), 9:1034-1048, Braasch, D. A., et al.,. Biochemistry (2003),42:7967-7975, Czauderna, F., et al., Nucleic Acids Research (2003),1:2705-2716, and McSwiggen et al., WO 03070918. However, no rationale isgiven in the prior art for the incorporation of 2′-modified nucleotides,nor any algorithm for the design of nuclease resistant dsRNAs, otherthan the modification of every, or every other, or of everypyrimidine-comprising, nucleotide in a dsRNA. In addition, some reportsshow that the gene expression inhibiting activity of a dsRNA inmammalian cells is increasingly compromised by the incorporation ofincreasing numbers of 2′-modified nucleotides. This is consistent withstudies in Drosophila melanogaster, which show that the completesubstitution of all nucleotides with 2′-O-alkyl modifications abolishedRNA interference activity (see Elbashir, S. M., et al., EMBO J. (2001)20:6877-6888), as well as with the findings of the present inventors.Thus, there is a need for a design method yielding the sites ofmodifications with maximum effect in stabilizing a specific dsRNAtowards degradation, while minimizing the overall number ofmodifications in order to preserve activity. The identification of sitesparticularly prone to enzymatic cleavage by the instant inventorsenables such a method.

The present inventors have discovered that the location of a2′-modification within a dsRNA has a significant influence on itsstabilizing effect. As shown in the examples herein, the stabilizingeffect of the incorporation of a 2′-O-methyl uridine is sequencespecific, with the most significant effect being observed in the contextof a 5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide. Whereas the uridinesin a 5′-ua-3′ sequence context are vulnerable to endonucleolytic attack,the uridines in 5′-AU-3′ sequence context are not. Finally, as evidencedby the data presented herein, the 2′-modifications must be present inboth strands of the modified dsRNA for optimal nuclease resistance.

Similar findings were obtained for the dinucleotides5′-uridine-guanine-3′ (5′-ug-3′), 5′-cytidine-adenine-3′ (5′-ca-3′),5′-uridine-uridine-3′ (5′-uu-3′), 5′-cytidine-cytidine-3′ (5′-cc-3′),5′-uridine-cytidine-3′ (5′-uc-3′), and 5′-cytidine-uridine-3′(5′-cu-3′). Particularly, it was found that it was possible to design adsRNA with a desired degree of stability by stepwise modification ofuridines and cytidines present in the above sequence contexts, e.g, in agiven siRNA having low stability in biological samples, by replacing inone step all uridines in a 5′-ua-3′ sequence context by 2′-modifieduridines, and in an optional further step, replacing all uridines in a5′-ua-3′ sequence context and all cytidines in a 5′-ca-3′ sequencecontext by the respective 2′-modified nucleotides, and, in an optionalfurther step, replacing all uridines in a 5′-ua-3′ sequence context andall cytidines in a 5′-ca-3′ sequence context and all uridines in a5′-ug-3′ by the respective 2′-modified nucleotides, and, in an optionalfurther step, replacing all uridines in a 5′-ua-3′ sequence context andall cytidines in a 5′-ca-3′ sequence context and all uridines in a5′-ug-3′ and all uridines in a 5′-uu-3′ sequence context by therespective 2′-modified nucleotides, and, in an optional further step,replacing all uridines in a 5′-ua-3′ sequence context and all cytidinesin a 5′-ca-3′ sequence context and all uridines in a 5′-ug-3′ and alluridines in a 5′-uu-3′ sequence context and all cytidines in a 5′-cc-3′sequence context by the respective 2′-modified nucleotides, and, in anoptional further step, replacing all uridines in a 5′-ua-3′ sequencecontext and all cytidines in a 5′-ca-3′ sequence context and alluridines in a 5′-ug-3′ and all uridines in a 5′-uu-3′ sequence contextand all cytidines in a 5′-cc-3′ sequence context and all uridines in a5′-uc-3′ sequence context by the respective 2′-modified nucleotides,and, in an optional further step, replacing all uridines in a 5′-ua-3′sequence context and all cytidines in a 5′-ca-3′ sequence context andall uridines in a 5′-ug-3′ and all uridines in a 5′-uu-3′ sequencecontext and all cytidines in a 5′-cc-3′ sequence context and uridines ina 5′-uc-3′ sequence context and all cytodines in a 5′-cu-3′ by therespective 2′-modified nucleotides, wherein, after each step, thestability of the modified dsRNA(s) in biological samples is determined.

The modified dsRNA of the present invention may be further stabilized bythe presence of at least one phosphorothioate internucleoside linkage.The present inventors have discovered that replacing a phosphoruslinkage with a phosphorothioate bond further increases the nucleaseresistance of the modified dsRNA. Not all of the phosphodiester bonds ofthe dsRNA need be replaced with phosphorothioate linkages. Although thephosphorothiate bond(s) may be located anywhere within the modifieddsRNA, the placement of the phosphorothioate bonds at specific locationshas a substantial effect on stability. Specifically, the presentinventors have discovered that incorporation of a phosphorothioate bondnear the ends of the dsRNA, and particularly in the nucleotideoverhang(s), has the most pronounced effect on nuclease resistance.

Thus, in another embodiment, the modified dsRNA comprises at least onephosphorothioate linkage, and preferably at least one phosphorothioatelinkage on each strand. In a preferred embodiment, the dsRNA containsmultiple phosphorothioate linkages on both RNA strands. Thephosphorothioate linkage is preferably introduced at or near the ends ofthe modified dsRNA, and particularly in a nucleotide overhang. Moreover,when the modified dsRNA contains a string of contiguous ornon-contiguous 2′-modified nucleotides, the dsRNA preferably contains atleast one, and more preferably at least two, phosphorothioate linkageswithin the string of 2′-modified nucleotides.

In another embodiment, at least one end of the modified dsRNA is blunt.dsRNA with at least one blunt end show improved stability as compared todsRNA having two nucleotide overhangs. dsRNA with at least one blunt endshows greater in vivo stability (i.e., is more resistant to degradationin the blood, plasma, and cells). However, dsRNA having at least onenucleotide overhang have superior inhibitory properties than theirblunt-ended counterparts. The presence of only one nucleotide overhangstrengthens the interference activity of the dsRNA, without effectingits overall stability. dsRNA having only one overhang has provenparticularly effective in vivo (as well as in a variety of cells, andcell culture mediums), and are more stable than dsRNA having two bluntends. The single-stranded nucleotide overhang may be 1 to 20, preferably1 to 10, more preferably 1 to 5, and most preferably 1 or 2, nucleotidesin length. Preferably, the single-stranded overhang is located at the3′-end of the antisense RNA strand. When the nucleotide overhang is 2nucleotides in length, the sequence of the 3′-end of the antisense RNAstrand is preferably 5′-GC-3′ or 5′-CGC-3′. In another preferredembodiment, the nucleotide overhang is at the 3′-end of the antisenseRNA strand, and the 5′-end is blunt. In a particularly preferredembodiment, the sequence of the 3′-end of the antisense RNA strand ispreferably 5′-GC-3′ or 5′-CGC-3′, wherein the phosphodiester bondswithin and adjacent to the overhang are replaced with phosphorothioatelinkages.

As described in co-pending U.S. Application Ser. No. 60/479,354, filedJun. 18, 2003, which is hereby incorporated in its entirety, thepresence of a purine base on the nucleotide overhang immediatelyadjacent to the terminal base pair provides further resistance todegradation. Thus, the modified dsRNA comprises a nucleotide overhang,wherein the unpaired nucleotide adjacent to the terminal base pair is apurine base, such as guanine (G) or adenine (A).

In yet another embodiment, the modified dsRNA is chemically modified tofurther enhance its stability, i.e. increase resistance to nucleasedegradation and/or strand dissociation. In this embodiment, theintegrity of the duplex structure is strengthened by at least one, andpreferably two, chemical linkages. Chemical linking may be achieved byany of a variety of well-known techniques, for example by introducingcovalent, ionic or hydrogen bonds; hydrophobic interactions, van derWaals or stacking interactions; by means of metal-ion coordination, orthrough use of purine analogues. Preferably, the chemical groups thatcan be used to modify the dsRNA include, without limitation, methyleneblue; bifunctional groups, preferably bis-(2-chloroethyl)amine;N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. Inone preferred embodiment, the linker is a hexa-ethylene glycol linker.In this case, the dsRNA are produced by solid phase synthesis and thehexa-ethylene glycol linker is incorporated according to standardmethods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996)35:14665-14670). In a particular embodiment, the 5′-end of the antisenseRNA strand and the 3′-end of the sense RNA strand are chemically linkedvia a hexa-ethylene glycol linker. Moreover, at least one nucleotide maybe modified to form a locked nucleotide. A locked nucleotide contains amethylene bridge that connects the 2′-oxygen of ribose with the4′-carbon of ribose. Oligonucleotides containing the locked nucleotideare described in Koshkin, A. A., et al., Tetrahedron (1998), 54:3607-3630) and Obika, S. et al., Tetrahedron Lett. (1998), 39:5401-5404). Introduction of a locked nucleotide into an oligonucleotideimproves the affinity for complementary sequences and increases themelting temperature by several degrees (Braasch, D. A. and D. R. Corey,Chem. Biol. (2001), 8:1-7).

In still another embodiment, the present invention provides dsRNA havingnucleotide sequences that are substantially identical or complementaryto at least a portion of a target gene. 100% sequence identity, orcomplementarity, between the inhibitory dsRNA and the portion of thetarget gene is typically preferred. However, embodiments of theinvention include modified dsRNA having at least 70%, or 85%, or 90% or95% sequence identity/complemetarity to the target gene, as well asimproved resistance to enzymatic degradation and/or dissociation, areencompassed by the present invention.

3. Method of Identifying and/or Making Modified Dsrna Having ImprovedStability

The invention further relates to a method to identify a dsRNA withincreased stability in biological samples, comprising the steps of:

-   -   (a) synthesizing a first dsRNA of the invention and a second        dsRNA identical to the first dsRNA except that it does not        comprise the 2′-modified nucleotides of the dsRNA of the        invention, and    -   (b) determining the stability of said first and said second        dsRNA in a biological sample by contacting both under identical        conditions with the biological sample, and monitoring their        degradation,

whereby, where the first dsRNA is degraded less rapidly than the seconddsRNA, a dsRNA with increased stability in biological samples isidentified.

In order to determine the stability of a dsRNA in a biological sample,the dsRNA is first brought into contact with the biological sample forsome time, or for various lengths of time. Subsequently, it isdetermined to what extent the dsRNA is still present, preferably in itsfull length form, or at least in a form that remains biologicallyactive, i.e. able to inhibit the expression of its target gene.

To this purpose, the dsRNA may be analyzed while still in contact withthe biological sample, for example by using a dsRNA labeled with afluorescent or radioactive marker. Alternatively, the dsRNA and/or itsdegradation products are first isolated from the constituents of thebiological sample, e.g. by extraction, precipitation, or filtering, andsubsequently analyzed, for example, without limitation, by gelelectrophoresis, mass spectrometry, capillary electrophoresis, or anyother method known to the skilled person.

A number of non-limiting examples for methods to determine the stabilityof a given dsRNA in a biological sample are given hereinbelow.

The invention further relates to a method for making a dsRNA havingimproved stability in biological samples and hence improvedbioavailability. The modified dsRNA of the invention can be isolatedfrom cells, produced from a DNA template, or can be chemicallysynthesized using methods known in the art prior to alteration using themethods of the invention.

In one embodiment, the modified dsRNAs are chemically synthesized. Amethod for making a double-stranded RNA (dsRNA) with high stability inbiological samples for inhibiting the expression of a target genecomprising according to the instant invention comprises one or more ofthe steps of:

-   -   (a) selecting one or more nucleotide sequences of between 18 and        30 nucleotides in length from the nucleotide sequence of the        mRNA resulting from the transcription of the target gene; and    -   (b) synthesizing one or more dsRNAs, wherein one strand        comprises a sequence complementary to one of the nucleotide        sequences selected in a.; and    -   (c) testing said one or more dsRNAs for their capability to        inhibit the expression of the target gene in a biological        sample; and    -   (d) selecting one of the one or more dsRNAs of c. possessing the        capability to inhibit the expression of the target gene in a        biological sample; and    -   (e) in the dsRNA selected in (d), identifying in the nucleotide        sequences of the sense strand as well as the antisense strand        all occurrences of the dinucleotides 5′-ua-3′,5′-ca-3′,5′-ug-3′,        5′-uu-3′,5′-cc-3′,5′-uc-3′ and 5′-cu-3′.    -   (f) synthesizing a dsRNA, wherein the 5′-uridines and/or        5′-cytidines in all occurrences of at least one of the        dinucleotides identified in e. is replaced by a 2′-modified        uridine and/or cytidine, respectively.

5′-uridines and/or 5′-cytidines in at least two, three, four, five, ormore than five of the dinucleotides identified in (e) may be replaced by2′-modified uridines and/or cytidines, respectively. Four isparticularly preferred, and the replacement in sequence motifs5′-ua-3′,5′-ca-3′, 5′-ug-3′, and 5′-uu-3′ is most preferred.

Another embodiment of the instant invention is a method to increase thenuclease resistance of a double stranded RNA (dsRNA), comprising thesteps of

-   -   (a) identifying in the nucleotide sequences of the sense strand        as well as the antisense strand of the dsRNA one, preferably        more than one, more preferably all occurrences of the        dinucleotides        5′-ua-3′,5′-ca-3′,5′-ug-3′,5′-uu-3′,5′-cc-3′,5′-uc-3′ and        5′-cu-3; and    -   (b) replacing at least one of the 5′-uridines and/or        5′-cytidines in the dinucleotides identified in (a) with a        2′-modified uridine and/or cytidine, respectively.

5′-uridines and/or 5′-cytidines in at least two, three, four, five, ormore than five of the dinucleotides identified in (a) may be replaced by2′-modified uridines and/or cytidines, respectively. Four isparticularly preferred, and the replacement in sequence motifs5′-ua-3′,5′-ca-3′, 5′-ug-3′, and 5′-uu-3′ is most preferred.

The above methods may be carried out stepwise, for example wherein

-   -   (a) in one step, all uridines in a 5′-ua-3′ sequence context are        replaced by 2′-modified uridines in addition to replacements        performed in a previous step, and,    -   (b) in an optional further step, all uridines in a 5′-ua-3′        sequence context and all cytidines in a 5′-ca-3′ sequence        context are replaced by the respective 2′-modified nucleotides,        and,    -   (c) in an optional further step, all uridines in a 5′-ua-3′        sequence context and all cytidines in a 5′-ca-3′ sequence        context and all uridines in a 5′-ug-3′ are replaced by the        respective 2′-modified nucleotides, and,    -   (d) in an optional further step, all uridines in a 5′-ua-3′        sequence context and all cytidines in a 5′-ca-3′ sequence        context and all uridines in a 5′-ug-3′ and all uridines in a        5′-uu-3′ sequence context are replaced by the respective        2′-modified nucleotides, and    -   (e) in an optional further step, all uridines in a 5′-ua-3′        sequence context and all cytidines in a 5′-ca-3′ sequence        context and all uridines in a 5′-ug-3′ and all uridines in a        5′-uu-3′ sequence context and all cytidines in a 5′-cc-3′        sequence context are replaced by the respective 2′-modified        nucleotides, and,    -   (f) in an optional further step, all uridines in a 5′-ua-3′        sequence context and all cytidines in a 5′-ca-3′ sequence        context and all uridines in a 5′-ug-3′ and all uridines in a        5′-uu-3′ sequence context and all cytidines in a 5′-cc-3′        sequence context and all uridines in a 5′-uc-3′ sequence context        are replaced by the respective 2′-modified nucleotides, and,    -   (g) in an optional further step, all uridines in a 5′-ua-3′        sequence context and all cytidines in a 5′-ca-3′ sequence        context and all uridines in a 5′-ug-3′ and all uridines in a        5′-uu-3′ sequence context and all cytidines in a 5′-cc-3′        sequence context and uridines in a 5′-uc-3′ sequence context and        all cytodines in a 5′-cu-3′ are replaced by the respective        2′-modified nucleotides,

wherein at least one of steps (a) through (g) is actually performed andwherein, after each step, the stability of the modified dsRNA(s) inbiological samples is determined.

The skilled person will readily understand how to modify the abovemethods for dsRNAs lacking an occurrence of one of the above sequencecontexts.

Preferably, at least four of the steps (a) through (g) are actuallyperformed, and more preferably the steps (a), (b), (c), and (d) arefirst performed in this order. However, it is also within the scope ofthe instant invention to switch the various sequence motifs in the steps(a) through (g) of the stepwise methods according to the invention. Forexample, without limitation, rather than first replacing all uridines ina 5′-ua-3′ sequence context by 2′-modified uridines, then replacing alluridines in a 5′-ua-3′ sequence context and all cytidines in a 5′-ca-3′sequence context, and then replacing all uridines in a 5′-ua-3′ sequencecontext and all cytidines in a 5′-ca-3′ sequence context and alluridines in a 5′-ug-3′, the stepwise method could be performed by firstreplacing all cytidines in a 5′-ca-3′ sequence context, then replacingall cytidines in a 5′-ca-3′ sequence context and all 5′-most uridines in5′-uu-3′ sequence context, and finally replacing all 5′-ca-3′ and all5′-most uridines in 5′-uu-3′ sequence context and all uridines in a5′-ug-3′ sequence context.

In general, the oligonucleotides of the present invention can besynthesized using protocols known in the art, for example, as describedin Caruthers, et al., Methods in Enzymology (1992) 211:3-19; Thompson,et al., International PCT Publication No. WO 99/54459; Wincott, et al.,Nucl. Acids Res. (1995) 23:2677-2684; Wincott, et al., Methods Mol.Bio., (1997) 74:59; Brennan, et al., Biotechnol. Bioeng. (1998)61:33-45; and Brennan, U.S. Pat. No. 6,001,11; each of which is herebyincorporated by reference in its entirety herein. In general, thesynthesis of oligonucleotides involves conventional nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-end,and phosphoramidites at the 3′-end. In a non-limiting example, smallscale syntheses are conducted on a Expedite 8909 RNA synthesizer sold byApplied Biosystems, Inc. (Weiterstadt, Germany), using ribonucleosidephosphoramidites sold by ChemGenes Corporation (Ashland, Mass., USA).Alternatively, syntheses can be performed on a 96-well platesynthesizer, such as the instrument produced by Protogene (Palo Alto,Calif., USA), or by methods such as those described in Usman, et al., J.Am. Chem. Soc. (1987) 109:7845; Scaringe, et al., Nucl. Acids Res.(1990) 18:5433; Wincott, et al., Nucl. Acids Res. (1990) 23:2677-2684;and Wincott, et al., Methods Mol. Bio. (1997) 74:59, each of which ishereby incorporated by reference in its entirety.

The nucleic acid molecules of the present invention may be synthesizedseparately and joined together post-synthetically, for example, byligation (Moore, et al., Science (1992) 256:9923; Draper, et al.,International PCT publication No. WO 93/23569; Shabarova, et al., Nucl.Acids Res. (1991) 19:4247; Bellon, et al., Nucleosides & Nucleotides(1997) 16:951; and Bellon, et al., Bioconjugate Chem. (1997) 8:204; orby hybridization following synthesis and/or deprotection. The nucleicacid molecules can be purified by gel electrophoresis using conventionalmethods or can be purified by high pressure liquid chromatography (HPLC;see Wincott et al., supra, the totality of which is hereby incorporatedherein by reference) and re-suspended in water.

In one embodiment, the method of making a modified dsRNA includessubstituting at least one uridine or cytidine phosphoramidite with thecorresponding 2′-O-methyl uridine or 2′-O-methyl cytidinephosphoramidite. In this embodiment, standard uridine or cytidinephosphoramidites are replaced with 2′-O-methyl uridine or 2′-O-methylcytidine phosphoramidites during the synthetic process. Phosphorothioatedinucleotide linkages are introduced by substituting an iodine oxidizersolution with a solution of the Beaucage reagent (J. Am. Chem. Soc.(1990) 12:1253) or, for example, with EDITH(3-ethoxy-1,2,4-dithiazoline-5-one; Xu et al., Nucleic Acids Research1996, 24:3643-3644). In addition, additional nucleotides containing Gand C bases may be inserted so as to produce appropriately placed G-Cbase pairs, i.e., positioned to form a terminal nucleotide base pairs orto produce G-C base pairs within the four consecutive terminalnucleotides of the duplex structure.

In another embodiment, at least one nucleotide of the dsRNA ischemically modified to introduce chemical moieties or other structuralfeatures that differ from those seen in naturally occurring RNA. Suchmodifications may affect the ability of a base to hydrogen bond with itsnormal complementary base, and include, without limitation, heterocyclicderivatives, nucleotide analogs, covalent modifications such as theintroduction of modified nucleotides, or the inclusion of pendant groupsthat are not naturally found in RNA molecules. Exemplary modificationsand methods for introducing such modifications into dsRNA are known inthe art, including those modifications and methods discussed in SectionII above and the references cited therein.

In other embodiments, dsRNA is isolated from cells or produced from aDNA template prior to alteration using methods known in the art. Inthese alternate embodiments, the stability of the dsRNA can be increasedprior to use by any of a number of well-known techniques, includingthose discussed above.

4. Methods to Inhibit the Expression of a Gene

In another aspect, the present invention relates to a method forinhibiting the expression of a target gene in a cell, comprising thesteps of introducing a dsRNA of the invention into the cell, andmaintaining the cell for a time sufficient to obtain inhibition ofexpression of the target gene in the cell, wherein methods of treatmentor diagnosis to be performed on a human or animal body are excluded. Thecell may be a mammalian cell.

The dsRNA may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing an organism in a solutioncontaining the dsRNA. Methods for oral introduction include directmixing of the dsRNA with food of the organism. Physical methods ofintroducing nucleic acids, for example, injection directly into the cellor extracellular injection into the organism, may also be used. Physicalmethods of introducing nucleic acids include injection of a solutioncontaining the dsRNA, bombardment by particles covered by the dsRNA,soaking the cell or organism in a solution of the dsRNA, orelectroporation of cell membranes in the presence of the dsRNA. Othermethods known in the art for introducing nucleic acids to cells may beused, such as lipid-mediated carrier transport, chemical-mediatedtransport, such as calcium phosphate, and the like. Thus the dsRNA maybe introduced along with components that perform one or more of thefollowing activities: enhance dsRNA uptake by the cell, stabilize theannealed strands, or otherwise increase inhibition of the target gene.

The present invention may be used to reduce the activity of the targetgene in a cell, for example, without limitation, for the analysis of thefunction of the target gene. Alternatively, the method may be used totest the effect of a second agent, e.g a small molecule, co-administeredwith the dsRNA, on the cell, under conditions of reduced expression ofthe target cell. In another embodiment, the invention is used forcosmetic purposes, for example to reduce the expression of a genepromoting unwanted hair growth, hair loss, coloring of the skin, callousformation, or to reduce the expression of a gene preventing or reducingmuscle formation.

5. Pharmaceutical Compositions Comprising dsRNA

As used herein, the term “treatment” refers to the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disorder, e.g., a disease or condition, asymptom of disease, or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve, or affect the disease, the symptoms of disease, or thepredisposition toward disease.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier ordiluent for administration of a therapeutic agent. Pharmaceuticallyacceptable carriers for therapeutic use are well known in thepharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, ed. 1985),which is hereby incorporated by reference herein. Such carriers include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The term specificallyexcludes cell culture medium. For drugs administered orally,pharmaceutically acceptable carriers include, but are not limited topharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid, or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

In one embodiment, the invention relates to a pharmaceutical compositioncomprising a modified dsRNA, as described in the preceding sections, anda pharmaceutically acceptable carrier, as described below. Apharmaceutical composition including the modified dsRNA is useful fortreating a disease caused by expression of a target gene. In this aspectof the invention, the dsRNA of the invention is formulated as describedbelow. The pharmaceutical composition is administered in a dosagesufficient to inhibit expression of the target gene.

The pharmaceutical compositions of the present invention areadministered in dosages sufficient to inhibit the expression or activityof the target gene. Compositions comprising the dsRNA of the inventioncan be administered at surprisingly low dosages. A maximum dosage of 5mg dsRNA per kilogram body weight per day may be sufficient to inhibitor completely suppress the expression or activity of the target gene.

In general, a suitable dose of modified dsRNA will be in the range of0.01 to 5.0 milligrams per kilogram body weight of the recipient perday, preferably in the range of 0.1 to 2.5 milligrams per kilogram bodyweight of the recipient per day, more preferably in the range of 0.1 to200 micrograms per kilogram body weight per day, and most preferably inthe range of 0.1 to 100 micrograms per kilogram body weight per day. Thepharmaceutical composition may be administered once per day, or thedsRNA may be administered as two, three, four, five, six or moresub-doses at appropriate intervals throughout the day. In that case, thedsRNA contained in each sub-dose must be correspondingly smaller inorder to achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of thedsRNA over a several day period. Sustained release formulations are wellknown in the art. In this embodiment, the dosage unit contains acorresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the infection or disease, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNA encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases. For example, mouse repositories canbe found at The Jackson Laboratory, Charles River Laboratories, Taconic,Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Networkand at the European Mouse Mutant Archive. Such models may be used for invivo testing of dsRNA, as well as for determining a therapeuticallyeffective dose.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), ocular,rectal, vaginal and topical (including buccal and sublingual)administration. In preferred embodiments, the pharmaceuticalcompositions are administered by intravenous or intraparenteral infusionor injection. The pharmaceutical compositions can also be administeredintraparenchymally, intrathecally, and/or by stereotactic injection.

For oral administration, the dsRNA useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredients mixed withpharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredient is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the pharmaceutical compositions of the invention will generally beprovided in sterile aqueous solutions or suspensions, buffered to anappropriate pH and isotonicity. Suitable aqueous vehicles includeRinger's solution and isotonic sodium chloride. In a preferredembodiment, the carrier consists exclusively of an aqueous buffer. Inthis context, “exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of dsRNA inthe cells that harbor the target gene or virus. Such substances include,for example, micellar structures, such as liposomes or capsids, asdescribed below. Although microinjection, lipofection, viruses, viroids,capsids, capsoids, or other auxiliary agents are required to introducedsRNA into cell cultures, surprisingly these methods and agents are notnecessary for uptake of dsRNA in vivo. The dsRNA of the presentinvention are particularly advantageous in that they do not require theuse of an auxiliary agent to mediate uptake of the dsRNA into the cell,many of which agents are toxic or associated with deleterious sideeffects. Aqueous suspensions according to the invention may includesuspending agents such as cellulose derivatives, sodium alginate,polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such aslecithin. Suitable preservatives for aqueous suspensions include ethyland n-propyl p-hydroxybenzoate.

The pharmaceutical compositions can also include encapsulatedformulations to protect the dsRNA against rapid elimination from thebody, such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811; PCT publication WO 91/06309; and European patent publicationEP-A-43075, which are incorporated by reference herein.

Toxicity and therapeutic efficacy of dsRNA can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.dsRNAs that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosages ofcompositions of the invention are preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anydsRNA used in the method of the invention, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range of the dsRNA or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test dsRNA which achieves a half-maximal inhibitionof symptoms) as determined in cell culture. Such information can be usedto more accurately determine useful doses in humans. Levels in plasmamay be measured, for example, by high performance liquid chromatography.

In addition to their administration individually or as a plurality, asdiscussed above, dsRNAs relating to the invention can be administered incombination with other known agents effective in treating viralinfections and diseases. In any event, the administering physician canadjust the amount and timing of dsRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

For oral administration, the dsRNA useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

6. Methods for Treating Diseases Caused by Expression of a Target Gene

In one embodiment, the invention relates to a method for treating asubject having a disease or at risk of developing a disease caused bythe expression of a target gene. In this embodiment, dsRNAs can act asnovel therapeutic agents for controlling one or more of cellularproliferative and/or differentiative disorders, disorders associatedwith bone metabolism, immune disorders, hematopoietic disorders,cardiovascular disorders, liver disorders, viral diseases, or metabolicdisorders. The method comprises administering a pharmaceuticalcomposition of the invention to the patient (e.g., a human), such thatexpression of the target gene is silenced. Because of their highefficiency and specificity, the dsRNA of the present inventionspecifically target mRNA of target genes of diseased cells and tissues,as described below, and at surprisingly low dosages. The pharmaceuticalcompositions are formulated as described in the preceding section, whichis hereby incorporated by reference herein.

Examples of genes which can be targeted for treatment include, withoutlimitation, an oncogene (Hanahan, D. and R. A. Weinberg, Cell (2000)100:57; and Yokota, J., Carcinogenesis (2000) 21(3):497-503); a cytokinegene (Rubinstein, M., et al., Cytokine Growth Factor Rev. (1998)9(2):175-81); a idiotype (Id) protein gene (Benezra, R., et al.,Oncogene (2001) 20(58):8334-41; Norton, J. D., J. Cell Sci. (2000)113(22):3897-905); a prion gene (Prusiner, S. B., et al., Cell (1998)93(3):337-48; Safar, J., and S. B. Prusiner, Prog. Brain Res. (1998)117:421-34); a gene that expresses molecules that induce angiogenesis(Gould, V. E. and B. M. Wagner, Hum. Pathol. (2002) 33(11):1061-3);adhesion molecules (Chothia, C. and E. Y. Jones, Annu. Rev. Biochem.(1997) 66:823-62; Parise, L. V., et al., Semin. Cancer Biol. (2000)10(6):407-14); cell surface receptors (Deller, M. C., and Y. E. Jones,Curr. Opin. Struct. Biol. (2000) 10(2):213-9); genes of proteins thatare involved in metastasizing and/or invasive processes (Boyd, D.,Cancer Metastasis Rev. (1996) 15(1):77-89; Yokota, J., Carcinogenesis(2000) 21(3):497-503); genes of proteases as well as of molecules thatregulate apoptosis and the cell cycle (Matrisian, L. M., Curr. Biol.(1999) 9(20):R776-8; Krepela, E., Neoplasma (2001) 48(5):332-49; Basbaumand Werb, Curr. Opin. Cell Biol. (1996) 8:71-738; Birkedal-Hansen, etal., Crit. Rev. Oral Biol. Med. (1993) 4:197-250; Mignatti and Rifkin,Physiol. Rev. (1993) 73:161-195; Stetler-Stevenson, et al., Annu. Rev.Cell Biol. (1993) 9:541-573; Brinkerhoff, E., and L. M. Matrisan, NatureReviews (2002) 3:207-214; Strasser, A., et al., Annu. Rev. Biochem.(2000) 69:217-45; Chao, D. T. and S. J. Korsmeyer, Annu. Rev. Immunol.(1998) 16:395-419; Mullauer, L., et al., Mutat. Res. (2001)488(3):211-1; Fotedar, R., et al., Prog. Cell Cycle Res. (1996)2:147-63; Reed, J. C., Am. J. Pathol. (2000) 157(5):1415-30; D'Ari, R.,Bioassays (2001) 23(7):563-5); genes that express the EGF receptor;Mendelsohn, J. and J. Baselga, Oncogene (2000) 19(56):6550-65; Normanno,N., et al., Front. Biosci. (2001) 6:D685-707); and the multi-drugresistance 1 gene, MDR1 gene (Childs, S., and V. Ling, Imp. Adv. Oncol.(1994) 21-36).

In the prevention of disease, the target gene may be one which isrequired for initiation or maintenance of the disease, or which has beenidentified as being associated with a higher risk of contracting thedisease. In the treatment of disease, the dsRNA can be brought intocontact with the cells or tissue exhibiting the disease. For example,dsRNA substantially identical to all or part of a mutated geneassociated with cancer, or one expressed at high levels in tumor cells,may be brought into contact with or introduced into a cancerous cell ortumor gene.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., a carcinoma, sarcoma, metastatic disorder orhematopoietic neoplastic disorder, such as a leukemia. A metastatictumor can arise from a multitude of primary tumor types, including butnot limited to those of prostate, colon, lung, breast and liver origin.As used herein, the terms “cancer,” “hyperproliferative,” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. These terms are meant to include all types ofcancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. Proliferative disordersalso include hematopoietic neoplastic disorders, including diseasesinvolving hyperplastic/neoplastic cells of hematopoietic origin, e.g.,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof.

The pharmaceutical compositions of the present invention can also beused to treat a variety of immune disorders, in particular thoseassociated with overexpression or aberrant expression of a gene orexpression of a mutant gene. Examples of hematopoietic disorders ordiseases include, without limitation, autoimmune diseases (including,for example, diabetes mellitus, arthritis (including rheumatoidarthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriaticarthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis,systemic lupus erythematosis, automimmune thyroiditis, dermatitis(including atopic dermatitis and eczematous dermatitis), psoriasis,Sjogren's Syndrome, Crohn's disease, aphthous ulcer, iritis,conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, drug eruptions, leprosy reversal reactions, erythema nodosumleprosum, autoimmune uveitis, allergic encephalomyelitis, acutenecrotizing hemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing, loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis,uveitis posterior, and interstitial lung fibrosis), graft-versus-hostdisease, cases of transplantation, and allergy.

In another embodiment, the invention relates to methods for treatingviral diseases, including but not limited to hepatitis C, hepatitis B,herpes simplex virus (HSV), HIV-AIDS, poliovirus, and smallpox virus.dsRNA of the invention are prepared as described herein to targetexpressed sequences of a virus, thus ameliorating viral activity andreplication. The dsRNAs can be used in the treatment and/or diagnosis ofviral infected tissue, both animal and plant. Also, such dsRNA can beused in the treatment of virus-associated carcinoma, such ashepatocellular cancer.

For example, the dsRNA of the present invention are useful for treatinga subject having an infection or a disease associated with thereplication or activity of a (+) strand RNA virus having a 3′-UTR, suchas HCV. In this embodiment, the dsRNA can act as novel therapeuticagents for inhibiting replication of the virus. The method comprisesadministering a pharmaceutical composition of the invention to thepatient (e.g., a human), such that viral replication is inhibited.Examples of (+) strand RNA viruses which can be targeted for inhibitioninclude, without limitation, picornaviruses, caliciviruses, nodaviruses,coronaviruses, arteriviruses, flaviviruses, and togaviruses. Examples ofpicornaviruses include enterovirus (poliovirus 1), rhinovirus (humanrhinovirus 1A), hepatovirus (hepatitis A virus), cardiovirus(encephalomyocarditis virus), aphthovirus (foot-and-mouth disease virusO), and parechovirus (human echovirus 22). Examples of calicivirusesinclude vesiculovirus (swine vesicular exanthema virus), lagovirus(rabbit hemorrhagic disease virus), “Norwalk-like viruses” (Norwalkvirus), “Sapporo-like viruses” (Sapporo virus), and “hepatitis E-likeviruses” (hepatitis E virus). Betanodavirus (striped jack nervousnecrosis virus) is the representative nodavirus. Coronaviruses includecoronavirus (avian infections bronchitis virus) and torovirus (Bernevirus). Arterivirus (equine arteritis virus) is the representativearteriviridus. Togavirises include alphavirus (Sindbis virus) andrubivirus (Rubella virus). Finally, the flaviviruses include flavivirus(Yellow fever virus), pestivirus (bovine diarrhea virus), andhepacivirus (hepatitis C virus). In a preferred embodiment, the virus ishepacivirus, the hepatitis C virus. Although the foregoing listexemplifies vertebrate viruses, the present invention encompasses thecompositions and methods for treating infections and diseases caused byany (+) strand RNA virus having a 3′-UTR, regardless of the host. Forexample, the invention encompasses the treatment of plant diseasescaused by sequiviruses, comoviruses, potyviruses, sobemovirus,luteoviruses, tombusviruses, tobavirus, tobravirus, bromoviruses, andclosteroviruses.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limitedto, oral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), ocular,rectal, vaginal, and topical (including buccal and sublingual)administration. In preferred embodiments, the pharmaceuticalcompositions are administered by intravenous or intraparenteral infusionor injection. The pharmaceutical compositions can also be administeredintraparenchymally, intrathecally, and/or by stereotactic injection.

7. Methods for Inhibiting Expression of a Target Gene

In yet another aspect, the invention relates to a method for inhibitingthe expression of a target gene in a cell or organism. In oneembodiment, the method comprises administering the inventive dsRNA or apharmaceutical composition comprising the dsRNA to a cell or anorganism, such as a mammal, such that expression of the target gene issilenced. Because of their surprisingly improved stability andbioavailability, the dsRNA of the present invention effectively inhibitexpression or activity of target genes at surprisingly low dosages.Compositions and methods for inhibiting the expression of a target geneusing dsRNA can be performed as described in the preceding sections,particularly Sections 4 and 5.

In this embodiment, a pharmaceutical composition comprising the dsRNAmay be administered by any means known in the art including, but notlimited to oral or parenteral routes, including intravenous,intramuscular, intraperitoneal, subcutaneous, transdermal, airway(aerosol), ocular, rectal, vaginal, and topical (including buccal andsublingual) administration. In preferred embodiments, the pharmaceuticalcompositions are administered by intravenous or intraparenteral infusionor injection. The pharmaceutical compositions can also be administeredintraparenchymally, intrathecally, and/or by stereotactic injection.

The methods for inhibiting the expression of a target gene can beapplied to any gene one wishes to silence, thereby specificallyinhibiting its expression, provided the cell or organism in which thetarget gene is expressed comprises the cellular machinery which effectsRNA interference. Examples of genes which can be targeted for silencinginclude, without limitation, developmental genes including but notlimited to adhesion molecules, cyclin kinase inhibitors, Wnt familymembers, Pax family members, Winged helix family members, Hox familymembers, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, andneurotransmitters and their receptors; (2) oncogenes including but notlimited to ABLI, BCL1, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2,ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2,MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TALI, TCL3 andYES; (3) tumor suppresser genes including but not limited to APC, BRCA1,BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53 and WT1; and (4) enzymesincluding but not limited to ACP desaturases and hydroxylases,ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases,amyloglucosidases, catalases, cellulases, cyclooxygenases,decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases,glucanases, glucose oxidases, GTPases, helicases, hemicellulases,integrases, invertases, isomerases, kinases, lactases, lipases,lipoxygenases, lysozymes, pectinesterases, peroxidases, phosphatases,phospholipases, phosphorylases, polygalacturonases, proteinases andpeptideases, pullanases, recombinases, reverse transcriptases,topoisomerases, and xylanases.

In addition to in vivo gene inhibition, the skilled artisan willappreciate that the dsRNA of the present invention are useful in a widevariety of in vitro applications. Such in vitro applications, include,for example, scientific and commercial research (e.g., elucidation ofphysiological pathways, drug discovery and development), and medical andveterinary diagnostics. In general, the method involves the introductionof the dsRNA into a cell using known techniques (e.g., absorptionthrough cellular processes, or by auxiliary agents or devices, such aselectroporation and lipofection), then maintaining the cell for a timesufficient to obtain degradation of an mRNA transcript of the targetgene.

8. Methods for Identifying Dsrna Having Increased Stability

In yet another aspect, the invention relates to methods for identifyingdsRNA having increased stability in biological tissues and fluids suchas serum. dsRNA having increased stability have enhanced resistance todegradation, e.g., by chemicals or nucleases (particularlyendonucleases) which normally degrade RNA molecules. Methods fordetecting increases in nucleic acid stability are well known in the art.Any assay capable of measuring or detecting differences between a testdsRNA and a control dsRNA in any measurable physical parameter may besuitable for use in the methods of the present invention. In general,because the inhibitory effect of a dsRNA on a target gene activity orexpression requires that the molecule remain intact, the stability of aparticular dsRNA can be evaluated indirectly by observing or measuring aproperty associated with the expression of the gene. Thus, the relativestability of a dsRNA can be determined by observing or detecting (1) anabsence or observable decrease in the level of the protein encoded bythe target gene, (2) an absence or observable decrease in the level ofmRNA product from the target gene, and (3) a change or loss in phenotypeassociated with expression of the target gene. In the context of amedical treatment, the stability of a dsRNA may be evaluated based onthe degree of the inhibition of expression or function of the targetgene, which in turn may be assessed based on a change in the diseasecondition of the patient, such as reduction in symptoms, remission, or achange in disease state.

In one embodiment, the method comprises preparing a dsRNA as describedin Section III above (e.g., through chemical synthesis), incubating thedsRNA with a biological sample, then analyzing and identifying thosedsRNA that exhibit an increased stability as compared to a controldsRNA.

In an exemplified embodiment, dsRNA is produced in vitro bymixing/annealing complementary single-stranded RNA strands, preferablyin a molar ratio of at least about 3:7, more preferably in a molar ratioof about 4:6, and most preferably in essentially equal molar amounts(e.g., a molar ratio of about 5:5). Preferably, the single-stranded RNAstrands are denatured prior to mixing/annealing, and the buffer in whichthe mixing/annealing reaction takes place contains a salt, preferablypotassium chloride. Single-stranded RNA strands may be synthesized bysolid phase synthesis using, for example, an Expedite 8909 synthesizer(Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany), asdescribed above.

dsRNA are incubated with a biological sample under the conditionssufficient or optimal for enzymatic function. After incubating with abiological sample, the stability of the dsRNA is analyzed by meansconventional in the art, for example using RNA gel electrophoresis asexemplified herein. For example, when the sample is serum, the dsRNA maybe incubated at a concentration of 1-10 μM, preferably 2-8 μM, morepreferably 3-6 μM, and most preferably 4-5 μM. The incubationtemperature is preferably between 25° C. and 45° C., more preferablybetween 35° C. and 40° C., and most preferably about 37° C.

The biological sample used in the incubation step may be derived fromtissues, cells, biological fluids or isolates thereof. For example, thebiological sample may be isolated from a subject, such as a wholeorganism or a subset of its tissues or cells. The biological sample mayalso be a component part of the subject, such as a body fluid, includingbut not limited to blood, serum, plasma, mucus, lymphatic fluid,synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amnioticcord blood, urine, vaginal fluid and semen. Preferably, the biologicalsample is a serum derived from a blood sample of a subject. The subjectis preferably a mammal, more preferably a human or a mouse.

In another embodiment, the method includes selecting a dsRNA havingincreased stability by measuring the mRNA and/or protein expressionlevels of a target gene in a cell following introduction of the dsRNA.In this embodiment, a dsRNA of the invention inhibits expression of atarget gene in a cell, and thus the method includes selecting a dsRNAthat induces a measurable reduction in expression of a target gene ascompared to a control dsRNA. Assays that measure gene expression bymonitoring RNA and/or protein levels can be performed within about 24hours following uptake of the dsRNA by the cell. For example, RNA levelscan be measured by Northern blot techniques, RNAse Protection Assays, orQuality Control-PCR (QC-PCR) (including quantitative reversetranscription coupled PCR(RT-PCR)) and analogous methods known in theart. Protein levels can be assayed, for example, by Western blottechniques, flow cytometry, or reporter gene expression (e.g.,expression of a fluorescent reporter protein, such as green fluorescentprotein (GFP)). RNA and/or protein levels resulting from target geneexpression can be measured at regular time intervals followingintroduction of the test dsRNA, and the levels are compared to thosefollowing introduction of a control dsRNA into cells. A control dsRNAcan be a nonsensical dsRNA (i.e., a dsRNA having a scrambled sequencethat does not target any nucleotide sequence in the subject), a dsRNAthat can target a gene not present in the subject (e.g., a luciferasegene, when the dsRNA is tested in human cells), or a dsRNA otherwisepreviously shown to be ineffective at silencing the target gene. ThemRNA and protein levels of the test sample and the control sample can becompared. The test dsRNA is selected as having increased stability whenthere is a measurable reduction in expression levels followingabsorption of the test dsRNA as compared to the control dsRNA. mRNA andprotein measurements can be made using any art-recognized technique(see, e.g., Chiang, M. Y., et al., J. Biol. Chem. (1991) 266:18162-71;Fisher, T, et al., Nucl. Acids Res. (1993) 21:3857; and Chen et al., J.Biol. Chem. (1996) 271:28259).

The ability of a dsRNA composition of the invention to inhibit geneexpression can be measured using a variety of techniques known in theart. For example, Northern blot analysis can be used to measure thepresence of RNA encoding a target protein. The level of the specificmRNA produced by the target gene can be measured, e.g., using RT-PCR.Because dsRNA directs the sequence-specific degradation of endogenousmRNA through RNAi, the selection methods of the invention encompass anytechnique that is capable of detecting a measurable reduction in thetarget RNA. In yet another example, Western blots can be used to measurethe amount of target protein present. In still another embodiment, aphenotype influenced by the amount of the protein can be detected.Techniques for performing Western blots are well known in the art (see,e.g., Chen, et al., J. Biol. Chem. (1996) 271:28259).

When the target gene is to be silenced by a dsRNA that targets apromoter sequence of the target gene, the target gene can be fused to areporter gene, and reporter gene expression (e.g., transcription and/ortranslation) can be monitored. Similarly, when the target gene is to besilenced by a dsRNA that targets a sequence other than a promoter, aportion of the target gene (e.g., a portion including the targetsequence) can be fused with a reporter gene so that the reporter gene istranscribed. By monitoring a change in the expression of the reportergene in the presence of the dsRNA, it is possible to determine theeffectiveness of the dsRNA in inhibiting the expression of the reportergene. The expression levels of the reporter gene in the presence of thetest dsRNA versus a control dsRNA are then compared. The test dsRNA isselected as having increased stability when there is a measurablereduction in expression levels of the reporter gene as compared to thecontrol dsRNA. Examples of reporter genes useful for use in the presentinvention include, without limitation, those coding for luciferase, GFP,chloramphenicol acetyl transferase (CAT), β-galactosidase, and alkalinephosphatase. Suitable reporter genes are described, for example, inCurrent Protocols in Molecular Biology, John Wiley & Sons, New York(Ausubel, F. A., et al., eds., 1989); Gould, S. J., and S. Subramani,Anal. Biochem. (1988) 7:404-408; Gorman, C. M., et al., Mol. Cell. Biol.(1982) 2:1044-1051; and Selden, R., et al., Mol. Cell. Biol. (1986)6:173-179; each of which is hereby incorporated by reference.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES

1. dsRNA Synthesis

1.1 Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

1.2 siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

Cholesterol was conjugated to siRNA as illustrated in FIG. 1. For thesynthesis of these 3′-cholesterol-conjugated siRNAs, an appropriatelymodified solid support was used for RNA synthesis. The modified solidsupport was prepared as follows:

1.2.1 Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature untilcompletion of the reaction was ascertained by TLC. After 19 h thesolution was partitioned with dichloromethane (3×100 mL). The organiclayer was dried with anhydrous sodium sulfate, filtered and evaporated.The residue was distilled to afford AA (28.8 g, 61%).

1.2.23-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It wasthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.Completion of the reaction was ascertained by TLC. The reaction mixturewas concentrated under vacuum and ethyl acetate was added to precipitatediisopropyl urea. The suspension was filtered. The filtrate was washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer was dried over sodium sulfate and concentrated togive the crude product which was purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

1.2.3 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acidethyl ester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated under vacuum, water was addedto the residue, and the product was extracted with ethyl acetate. Thecrude product was purified by conversion into its hydrochloride salt.

1.2.43-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. Thesuspension was cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) wasadded. The reaction mixture was stirred overnight. The reaction mixturewas diluted with dichloromethane and washed with 10% hydrochloric acid.The product was purified by flash chromatography (10.3 g, 92%).

1.2.51-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD was added slowly with stirring within 20 mins. Thetemperature was kept below 5° C. during the addition. The stirring wascontinued for 30 mins at 0° C. and 1 mL of glacial acetic acid wasadded, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water Theresultant mixture was extracted twice with 100 mL of dichloromethaneeach and the combined organic extracts were washed twice with 10 mL ofphosphate buffer each, dried, and evaporated to dryness. The residue wasdissolved in 60 mL of toluene, cooled to 0° C. and extracted with three50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extractswere adjusted to pH 3 with phosphoric acid, and extracted with five 40mL portions of chloroform which were combined, dried and evaporated todryness. The residue was purified by column chromatography using 25%ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

1.2.6[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) was added, the mixture was extracted with ethylacetate (3×40mL). The combined ethylacetate layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which waspurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was carried out at room temperature overnight.The reaction was quenched by the addition of methanol. The reactionmixture was concentrated under vacuum and to the residue dichloromethane(50 mL) was added. The organic layer was washed with 1M aqueous sodiumbicarbonate. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine was removed byevaporating with toluene. The crude product was purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

1.2.7 Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3mL), triethylamine (0.18 g, 0.440 mL, 3.15 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 mL) and washed with icecold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step.

1.2.8 Cholesterol derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM)was added. The suspension was agitated for 2 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The achieved loading of the CPG was measured bytaking UV measurement (37 mM/g).

The synthesis and structure of cholesterol conjugated RNA strands isillustrated in FIG. 1.

2. siRNA Agent Design and Selection

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 1.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation^(a) Nucleotide(s) A, a2′-deoxy-adenosine-5′-monophosphate, adenosine-5′-monophosphate C, c2′-deoxy-cytidine-5′-monophosphate, cytidine-5′-monophosphate G, g2′-deoxy-guanosine-5′-monophosphate, guanosine-5′-monophosphate T, t2′-deoxy-thymidine-5′-monophosphate, thymidine-5′-monophosphate U, u2′-deoxy-uridine-5′-monophospate, uridine-5′-monophospate N, n any2′-deoxy-nucleotide/nucleotide (G, A, C, or T, g, a, c or u) am2′-O-methyladenosine-5′-monophospate cm2′-O-methylcytidine-5′-monophospate gm2′-O-methylguanosine-5′-monophospate tm2′-O-methyl-thymidine-5′-monophospate um2′-O-methyluridine-5′-monophospate af2′-fluoro-2′-deoxy-adenosine-5′-monophospate cf2′-fluoro-2′-deoxy-cytidine-5′-monophospate gf2′-fluoro-2′-deoxy-guanosine-5′-monophospate tf2′-fluoro-2′-deoxy-thymidine-5′-monophospate uf2′-fluoro-2′-deoxy-uridine-5′-monophospate A, C, G, T, U, a, underlined:nucleoside-5′-phosphorothioate c, g, t, u am, cm, gm, tm, underlined:2′-O-methyl-nucleoside-5′-phosphorothioate um

bold italic: 2′-deoxy-adenosine, 2′-deoxy-cytidine, 2′-deoxy-guanosine,2′-

deoxy-thymidine, 2′-deoxy-uridine, adenosine, cytidine, guanosine,thymidine, uridine (5′-hydroxyl)

bold italic: 2′-O-methyl-adenosine, 2′-O-methyl-cytidine, 2′-O-methyl-

guanosine, 2′-O-methyl-thymidine, 2′-O-methyl-uridine (5′-hydroxyl) -tp2′- or 3′-terminal phosphate or 2′/3′-terminal cyclic phosphate -Chol1-{6-[cholester-3-yloxycarbonylamino]-hexanoyl}-4-hydroxy-pyrrolidin-3-phosphorothioate diester ^(a)capital letters represent2′-deoxyribonucleotides (DNA), lower case letters representribonucleotides (RNA)

Table 2 summarizes the sequences of the sense and antisense strands of afirst group of double stranded RNAs tested for stability in human serumherein.

TABLE 2Sequences of sense and antisense strands of a first group of double stranded RNAstested for stability in human serum. All sequences are given 5′ → 3′.SEQ SEQ Duplex ID ID Descriptor Sense NO: Antisense NO: GE1s/GE1as

aucacccuccuuaaauauuu  2

ucuagugggaggaauuuauaaa  3 GE7s/GE7as

aumcacccumccumumaaaumaumumum  4

gcumagumgggaggaaumumumaumaaa  5 GE7s/GE8as

aumcacccumccumumaaaumaumumum  4

gmcmumagumgggaggaaumumumaumaaa  6 GE7s/GE9as

aumcacccumccumumaaaumaumumum  4

gmcmumagumgggaggaaumumumaumaaa  7 GE7s/GE10as

aumcacccumccumumaaaumaumumum  4

gmcmumagugggaggaauuumaumaaa  8 GE7s/GE11as

aumcacccumccumumaaaumaumumum  4

gmcmuagugggaggaaumuuaumaaa  9 GE1s/GE7as

aucacccuccuuaaauauuu  2

gcumagumgggaggaaumumumaumaaa  5 GE1s/GE8as

aucacccuccuuaaauauuu  2

gmcmumagumgggaggaaumumumaumaaa  6 GE1s/GE9as

aucacccuccuuaaauauuu  2

gmcmumagumgggaggaaumumumaumaaa  7 GE1s/GE10as

aucacccuccuuaaauauuu  2

gmcmumagugggaggaauuumaumaaa  8 GE1s/GE11as

aucacccuccuuaaauauuu  2

gmcmuagugggaggaaumuuaumaaa  9 GE7s/GE1as

aumcacccumccumumaaumaumumum  4

ucuagugggaggaauuuauaaa  3 LC1s/LC1as

uuacgcugaguacuucgaTT 10

cgaaguacucagcguaagTT 11 LC2s/LC2as

cggaucaaaccucaccaaTT 12

cgaaguacucagcguaagTT 13 AL-DP-5097

uuuacaagccuugguucagu 14

cugaaccaaggcuuguaaagug 15 AL-DP-5398

umumumacmaagccumumggumggumucmagu 16 acumgaaccmaaggcuumgumaaagumg 17AL-DP-5458

ufufufacfaagccufufggufucfagu 18 acufgaaccfaaggcuufgufaaagmumg 19AL-DP-5542

umumumacmaagccumumggumucmagu-Chol 20

cumgaaccmaaggcuumgumaaagmumg 21 AL-DP-5543

uuuacaagccuugguucagu-Chol 22

cugaaccaaggcuuguaaagmumg 23 AL-DP-5098

gaaucuuauauuugauccaa 24

uggaucaaauauaagauucccu 25 AL-DP-5399

gaaucumumaumaumumumgauccmaa 26 umumggaucmaaaumaumaagaumuccmcmu 27AL-DP-5459

gaaucufufaufaufufufgauccfaa 28 ufufggaucfaaaufaufaagaufuccmcmu 29AL-DP-5544

gaaucumumaumaumumumgauccmaa-Chol 30

umggaucmaaaumaumaagaumuccmcmu 31 AL-DP-5545

gaaucuuauauuugauccaa-Chol 32

uggaucaaauauaagauuccmcmu 33 AL-DP-5024

gguguauggcuucaacccug 34

aggguugaagccauacaccucu 35 AL-DP-5388

ggumgumaumggcumucmaacccug 36 cmagggumumgaagccmaumacmaccumcmu 37AL-DP-5448

ggufgufaufggcufucfaacccug 38 cfagggufufgaagccfaufacfaccumcmu 39AL-DP-5013

guguauggcuucaacccuga 40

caggguugaagccauacaccuc 41 AL-DP-5387

gumgumaumggcumucmaacccumga 42 ucmagggumumgaagccmaumacmaccmumc 43AL-DP-5447

gufgufaufggcufucfaacccufga 44 ucfagggufufgaagccfaufacfaccmumc 45AL-DP-5084

ugaacaucaagaggggcauc 46

augccccucuugauguucagga 47 AL-DP-5394

umgaacmaucaagaggggcmauc 48 gaumgccccucumumgaumgumucmagga 49 AL-DP-5454

ufgaacfaucaagaggggcfauc 50 gaufgccccucufufgaufgufucfagmgma 51 AL-DP-5094

ccccaucacuuuacaagccu 52

ggcuuguaaagugauggggcug 53 AL-DP-5397

ccccmaucmacumumumacmaagccu 54 aggcumumgumaaagumgaumggggcmumg 55AL-DP-5457

ccccfaufacufufufacfaagccu 56 aggcufufgufaaagufgaufggggcmumg 57AL-DP-5093

cacauccuccaguggcugaa 58

ucagccacuggaggaugugagu 59 AL-DP-5396

cmacmauccuccmagumggcumgaa 60 umucmagccmacumggaggaumgumgagu 61 AL-DP-5456

cfacfauccuccfagufggcufgaa 62 ufucfagccfacufggaggaufgufgamgmu 63AL-DP-5089

aguuugugacaaauaugggc 64

cccauauuugucacaaacucca 65 AL-DP-5395

agumumumgumgacmaaaumaumgggc 66 gcccaumaumumumgucmacmaaacucmcma 67AL-DP-5455

agufufufgufgacfaaaufaufgggc 68 gcccaufaufufufgucfacfaaacucmcma 69AL-DP-5030

aacaccaacuucuuccacga 70

cguggaagaaguugguguucau 71 AL-DP-5389

aacmaccmaacumucumuccmacga 72 ucgumggaagaagumumggumgumucmau 73 AL-DP-5449

aacfaccfaacufucufuccfacga 74 ucgufggaagaagufufggufgufucmamu 75AL-DP-5035

accaacuucuuccacgaguc 76

acucguggaagaaguugguguu 77 AL-DP-5390

accmaacumucumuccmacgaguc 78 gacucgumggaagaagumumggumgumu 79 AL-DP-5450

accfaacufucufuccfacgaguc 80 gacucgufggaagaagufufggumgmumu 81 AL-DP-5046

ucaagugucaucacacugaa 82

ucagugugaugacacuugauuu 83 AL-DP-5391

ucmaagumgucmaucmacmacumgaa 84 umucmagumgumgaumgacmacumumgaumumu 85AL-DP-5451

ucfaagufgucfaucfacfacufgaa 86 ufucfagufgufgaufgacfacufufgaumumu 87AL-DP-5048

ucaucacacugaauaccaau 88

uugguauucagugugaugacac 89 AL-DP-5392

ucmaucmacmacumgaaumaccmaau 90 aumumggumaumucmagumgumgaumgacmac 91AL-DP-5452

ucfaucfacfacufgaaufaccfaau 92 aufufggufaufucfagufgufgaufgacmamc 93AL-DP-5002

auugauugaccuguccauuc 94

aauggacaggucaaucaaucuu 95 AL-DP-5386

aumumgaumumgaccumguccmaumuc 96 gaaumggacmaggucmaaucmaaucmumu 97AL-DP-5446

aufufgaufufgaccufguccfaumuc 98 gaaufggacfaggucfaaucfaaucmumu 99AL-DP-5049

uguccauucaaaacuaccac 100

ugguaguuuugaauggacaggu 101 AL-DP-5393

umguccmaumucmaaaacumaccmac 102 gumggumagumumumumgaaumggacmaggu 103AL-DP-5453

ufguccfaufucfaaaacufaccfac 104 pgufggufagufufufufgaaufggacfagmgmu 105AL-DP-5437

ucaucacacugaauaccaau 106

uugguauucagugugaugacac 107 AL-DP-5392

ucmaucmacmacumgaaumaccmaau 108 paumumggumaumucmagumgumgaumgacmac 109AL-DP-HCV

cggcuagcugugaaaggucc 110

gaccuuucacagcuagccguga 111

The sense strands GE1s and GE7s were annealed respectively with theantisense strands GE1as, GE8 as, GE9 as, GE10as, GEl11as; sense strandLC1s was annealed with antisense strand LC1as. The resulting doublestrand RNA duplexes are referred to herein as [sense strand]/[antisensestrand], e.g. GE1s/GE1as is used to denominate the duplex formed byannealing of GE1s with GE1as.

The nucleotides at positions 1-21, counting 5′ to 3′, of the antisensestrands GE1as, GE7 as, GE8 as, GE9 as, GE10as, and GE11as arecomplementary to position 1488-1508 of the sequence available underGenBank accession number X75932. The nucleotides at positions 1-19 ofthe antisense strand LC1as are complementary to positions 434-452 of thesequence available under GenBank accession number U47298.

AL-DP-5097, AL-DP-5398, AL-DP-5458, AL-DP-5542, and AL-DP-5543 areidentical in sequence except for 2′-modifications, phosphorothioatelinkages, and 3′-teminal cholesteryl ligands. The antisense strands arecomplementary to positions 1259-1281 of the human ApoB gene (GenBankaccession no. NM_(—)000384).

AL-DP-5098, AL-DP-5399, AL-DP-5459, AL-DP-5544, and AL-DP-5545 areidentical in sequence except for 2′-modifications, phosphorothioatelinkages, and 3′-teminal cholesteryl ligands. The antisense strands arecomplementary to positions 2096-2118 of the human ApoB gene.

AL-DP-5024, AL-DP-5388, and AL-DP-5448 are identical in sequence exceptfor 2′-modifications, phosphorothioate linkages, and 3′-teminalcholesteryl ligands. The antisense strands are complementary topositions 428-450 of the human ApoB gene.

AL-DP-5013, AL-DP-5387, and AL-DP-5447 are identical in sequence exceptfor 2′-modifications, phosphorothioate linkages, and 3′-teminalcholesteryl ligands. The antisense strands are complementary topositions 429-451 of the human ApoB gene.

AL-DP-5084, AL-DP-5394, and AL-DP-5454 are identical in sequence exceptfor 2′-modifications, phosphorothioate linkages, and 3′-teminalcholesteryl ligands. The antisense strands are complementary topositions 586-608 of the human ApoB gene.

AL-DP-5094, AL-DP-5397, and AL-DP-5457 are identical in sequence exceptfor 2′-modifications, phosphorothioate linkages, and 3′-teminalcholesteryl ligands. The antisense strands are complementary topositions 1250-1272 of the human ApoB gene.

AL-DP-5093, AL-DP-5396, and AL-DP-5456 are identical in sequence exceptfor 2′-modifications, phosphorothioate linkages, and 3′-teminalcholesteryl ligands. The antisense strands are complementary topositions 1302-1324 of the human ApoB gene.

AL-DP-5089, AL-DP-5395, and AL-DP-5455 are identical in sequence exceptfor 2′-modifications, phosphorothioate linkages, and 3′-teminalcholesteryl ligands. The antisense strands are complementary topositions 2770-2792 of the human ApoB gene.

AL-DP-5030, AL-DP-5389, and AL-DP-5449 are identical in sequence exceptfor 2′-modifications, phosphorothioate linkages, and 3′-teminalcholesteryl ligands. The antisense strands are complementary topositions 2829-2851 of the human ApoB gene.

AL-DP-5035, AL-DP-5390, and AL-DP-5450 are identical in sequence exceptfor 2′-modifications, phosphorothioate linkages, and 3′-teminalcholesteryl ligands. The antisense strands are complementary topositions 2832-2854 of the human ApoB gene.

AL-DP-5046, AL-DP-5391, and AL-DP-5451 are identical in sequence exceptfor 2′-modifications, phosphorothioate linkages, and 3′-teminalcholesteryl ligands. The antisense strands are complementary topositions 10158-10180 of the human ApoB gene.

AL-DP-5048, AL-DP-5392, and AL-DP-5452 are identical in sequence exceptfor 2′-modifications, phosphorothioate linkages, and 3′-teminalcholesteryl ligands. The antisense strands are complementary topositions 10165-10187 of the human ApoB gene.

AL-DP-5002, AL-DP-5386, and AL-DP-5446 are identical in sequence exceptfor 2′-modifications, phosphorothioate linkages, and 3′-teminalcholesteryl ligands. The antisense strands are complementary topositions 13682-13704 of the human ApoB gene.

AL-DP-5049, AL-DP-5393, and AL-DP-5453 are identical in sequence exceptfor 2′-modifications, phosphorothioate linkages, and 3′-teminalcholesteryl ligands. The antisense strands are complementary topositions 13693-13715 of the human ApoB gene.

The sense strand of AL-DP-HCV corresponds to positions 9475-9495 of the3′-untranslated region of hepatitis C virus (GenBank accession number:D89815).

3. Serum Incubation Assay

Blood of 8 human volunteers (270 mL) was collected and kept at roomtemperature for 3 hours. The blood pool was then centrifuged at 20° C.and 3000 rcf using Megafuge 1.0 (Heraeus Instruments, Kendro LaboratoryProducts GmbH, Langenselbold) to separate the serum from the cellularfraction. The supernatant was stored in aliquots at −20° C. and used asneeded. Alternatively, human serum or mouse serum obtained from Sigma(Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany, cat. No. human serumH1513, mouse serum M5905) was employed. Assay results reported hereinwere consistent among the different human serum sources. Mouse serumshowed somewhat higher exonucleolytic activity than human serum.

Double stranded RNAs (300 μmol, ca. 4.2 μg) dissolved in 6 μl phosphatebuffered saline (PBS) were added to 60 μl human serum in a 1.5 mlEppendorf tube, and the mixture was incubated for varying extents oftime, e.g. 0, 15, or 30 minutes, or 1, 2, 4, 8, 16, or 24 hours in athermomixer (Eppendorf thermomixer comfort; Eppendorf, Hamburg, Germany)at 37° C. and 1050 rpm. Subsequently, the whole tube containing theRNA/serum solution was immediately processed further or frozen in liquidnitrogen and stored at −80° C. until analysis. As a control, the sameamount of double stranded RNA was added to 60 μl annealing buffer,incubated a 37° C. for 0, 24 or 48 hours, and immediately furtherprocessed or frozen in liquid nitrogen and stored at −80° C. untilanalysis.

4. Analysis by Electrophoresis and “Stains all” Detection

For analysis, frozen serum incubation samples from store were thawed,their constituents were isolated by phenol-extraction andethanol-precipitation, separated on denaturing 14% polyacrylamide gels(6M Urea, 20% formamide, Carl Roth GmbH & Co KG Karlsruhe, Germany) anddetected by staining with the “stains-all” reagent (Sigma-Aldrich ChemieGmbH, Steinheim, Germany). Reaction mixtures from incubation of selecteddsRNAs were further analysed covering the time points 8 hours, 16 hoursand 24 hours.

4.1 Isolation of siRNA

For analysis, the frozen samples were placed on ice and 450 μl of 0.5 MNaCl was added, followed by brief vortexing. After complete thawing, theresulting solution was transferred to Phase-Lock Gel tubes (Eppendorf,Hamburg, Germany; cat. No. 0032 005.152), fixed with 500 μl 50% phenol,48% chloroform, 2% isoamylacohol (Carl Roth GmbH & Co KG, Karlsruhe,Germany, cat. No. A156.2), and an additional 300 μl chloroform wereadded. The tubes were vortexed vigorously for 30 seconds andsubsequently centrifuged for 15 min at 16.200 rcf and 4° C. The aqueoussupernatant was transferred to a fresh tube, mixed with 40 μl MNa-acetate pH 5.2, 1 μl GlycoBlue (Ambion, Tex., USA; cat. No. 9515) and1 ml Ethanol 95%, and vortexed vigorously for ca. 20 s. Precipitation ofRNA was brought to completion over night at −20° C.

4.2 Denaturing Gel Electrophoresis and RNA Staining

The following day, tubes were centrifuged for 30 min at 16.200 rcf and4° C. The supernatant was removed and discarded. The RNA pellet waswashed with 500 μl ice-cold Ethanol 70%, and re-pelleted bycentrifugation for 10 min at 16.200 rcf and 4° C. All liquid wasremoved, the pellet was air-dried for 1 min and dissolved in 12 μl gelloading buffer (95% formamide, 5% EDTA 1M, 0.02% xylene cyanol, 0,02%bromophenol blue). The gel was pre-run for 1 h at 100 W. The sampleswere boiled for 10 min at 95° C. and chilled quickly on ice. 4 μl wereloaded on a denaturing 14% polyacrylamide gel (8M Urea, 20% formamide,19:1 acrylamide:N,N-Methylenebisacrylamide). The RNA was separated forabout 40 min at 100 W (Sequi-Gen GT gel system, 38×30 cm, Bio-RadLaboratories, Hercules, Calif., USA, cat. no. 165-3862). RNA bands werevisualized by staining with the “stains-all” reagent (Sigma-AldrichChemie GmbH, Steinheim, Germany, cat. no. E9379) according tomanufacturer's instructions.

5. Analysis by LC/MS

Frozen serum incubation samples from store were thawed, theirconstituents were isolated by phenol-extraction andethanol-precipitation, the resulting pellet was dissolved in 50 μl ofwater and 42 μl of this solution was injected into the HPLC-system(Amersham Biosciences Ettan mLC-System equipped with UV-detection systemand Jetstream column heater coupled to Thermo Finnigan LCQ DecaXp massspectrometer; HPLC column: Waters Xterra C8-MS; 2.1×50 mm; particle size2.5 μm; 60° C.; Flow 200 μl/min; UV-Detection at 254 nm; ESI source andIonTrap-detector, total ion current detection for MS-detection). TheMS-instrument was started with a 3 min time delay after the injection toprotect the mass spectrometer from salt and other unbound sampleimpurities. An eluent gradient was employed as follows:

Eluents:

A: 400 mM Hexafluoroisopropanol/16.3 mM Triethylamine in deionizedwater; pH=7.9

B: Methanol (LC-MS grade)

Gradient table: 0->3 min: 1% B; 3->33 min: 1%->24% B linear; 33.1 min:100% B; 33.1->35 min: 100% B 35.1 min: 1% B 35.1->38 min: 1% B

Due to the denaturing conditions on the column (60° C.), all samplefragments elute as single strands. Every fragment is detected as onepeak in the UV- and the TIC-chromatogram. A raw mass spectrum of everypeak is extracted from the TIC-chromatogram and deconvoluted using thesoftware “Bioworks”, Version 3.1 (Thermo Electron GmbH, Dreieich,Germany). At the end of each run, the column is washed andre-equilibrated.

Comparison between the experimentally detected and the calculated massesof all possible fragments of both duplex strands leads to theidentification of the cleavage fragments generated during serumincubation. The LC-MS method is insensitive to phosphate-induced shiftsas are the gel assays and allows an exact mapping of all cleavage sites.

Data evaluation steps:

1.) Average isotopic masses are calculated for all hypotheticalfragments of both the sense and the antisense strand.

2.) 62.0 Da are added to the hypothetical fragment masses to account fora cyclic diester phosphate terminus at the cleavage site; for a cyclicdiester phosphorothioate at the cleavage position, +78.06 Da are added;80.0 Da are added for a 2′- or 3′-monoester phosphate group, and +96.06Da are added for a phosphorothioate monoester.

3.) All experimental masses are compared to these predicted masses; afragment is identified if its experimentally determined mass falls towithin +/−1 Da of the predicted mass.

4.) Fragments unequivocally assignable allow the identification ofcleavage positions.

5.) The increase or decrease of a peak at different time points allowconclusions on whether the cleavage is a primary or secondary event.

2′-3′-cyclic phosphates and the resulting hydrolyzed 2′- or3′-phosphates as possible termini after the cleavage event were includedinto the mass calculation of the fragments. This end group analysisprovided an insight to the cleavage mechanism. If a cyclic phosphate wasdetected at the 3′-end of a fragment resulting from cleavage, anucleophilic attack of the 2′-OH on the 3′-O—P phosphorus of theinternal base must have been involved in the cleavage mechanism. Forstabilization against degradation following this mechanism, a2′-modification, e.g. a 2′-O-Methyl substitution on the respectivenucleotide, may be employed.

6. Determination of siRNA Degradation Time Course by HPLC FollowingProteinase K Treatment of Serum Samples

In order to get a more quantitative means of determining siRNA stranddegradation, a method comprising Proteinase K treatment of serum samplesfollowed by the separation of serum sample constituents on an HPLC wasdeveloped. By comparison of different modified and unmodified siRNAs,this method can be used to identify sites and sequence motifs in siRNAstrands that are particularly vulnerable to nucleolytic degradation.

The example below shows the analyses of serum samples which werecontacted with siRNAs in vitro. However, this method can equally beapplied to biological samples ex vivo, i.e. obtained from a subjectwhich was contacted with an siRNA in vivo.

Proteinase K (20 mg/ml) was obtained from peQLab (Erlangen, Germany;Cat.-No. 04-1075) and diluted 1:1 with deionized water (18.2 mΩ) to afinal concentration of 10 mg/ml Proteinase K. Proteinase K Buffer (4.0ml TRIS-HCl 1M pH 7.5, 1.0 ml EDTA 0.5M, 1.2 ml NaCl 5M, 4.0 ml SDS 10%)was prepared fresh and kept at 50° C. until use to avoid precipitation.

A 40 mer of poly(L-dT), (L-dT)₄₀ was obtained from Noxxon Pharma AG(Berlin, Germany) and used as an internal standard. Polymers of theL-enantiomers of nucleic acids show an extraordinary stability towardsnucleolytic degradation (Klussman S, et al., Nature Biotechn. 1996,14:1112) but otherwise very similar properties when compared tonaturally occurring nucleic acids consisting of R-enantiomers.

6.1 Proteinase K Treatment of Serum Incubation Samples

To terminate the siRNA-degradation, 25 μl of Proteinase K buffer wereadded to serum incubation samples immediately after expiry of therespective incubation period, the mixture vortexed at highest speed for5 s (Vortex Genie 2, Scientific Industries, Inc., Bohemia, N.Y., USA,cat. no. S10256), 8 μl Proteinase K (10 mg/ml) were added followed byvortexing for 5 s, and finally the mixture was incubated for 20 min in athermomixer at 42° C. and 1050 rpm.

6 μl of a 50 μM solution (300 pmole) of (L-dT)₄₀ were added as aninternal standard, the solution was vortexed for 5 s, and the tubecentrifuged for 1 min in a tabletop centrifuge to collect all dropletsclinging to the inner surfaces of the tube at the bottom. The solutionwas transferred to a Microcon Centrifugal Filter Unit YM-100 (MilliporeGmbH, Eschborn, Germany, Cat. No. 42413) and filtered by centrifugationat 21900 rcf for 45 min.

The incubation tube was washed with 47.5 μl deionized water (18.2 mg-2),the wash filtered through the Microcon Centrifugal Filter Unit at 21900rcf for 15 min, and the wash step repeated. Approximately 180 μl of thetheoretical total volume of 200 μl are on average recovered after thesecond washing step.

6.2 Ion Exchange Chromatographic Separation of siRNA Single Strands fromEach Other and From Degradation Products:

A Dionex BioLC HPLC-system equipped with inline-degasser, autosampler,column oven and fixed wavelength UV-detector (Dionex GmbH, Idstein,Germany) was used under denaturing conditions. Standard run parameterswere:

-   Column: Dionex DNA-Pac100; 4×250 mm-   Temperature: 75° C.-   Eluent A: 10 mM NaClO₄, 20 mM TRIS-HCl, 1 mM EDTA; 10% acetonitrile,    pH=8.0-   Eluent B: 800 mM NaClO₄, 20 mM TRIS-HCl, 1 mM EDTA; 10%    acetonitrile, pH=8.0-   Detection: @ 260 nm

Gradient:  0-1 min: 10% B  1-11 min: 10% -> 35% B 11-12 min: 35% B ->100% B 12-14 min: 100% B->10% B 14-16 min: 10% B for columnreequilibration

-   Injection volume: 20 μl

Where separation between the two strands of an siRNA was notsatisfactory or a degradation fragment co-eluted with one strand, thechromatographic parameters were adjusted by changing temperature, pH,and/or the concentration of acetonitrile, until separation was achievedwhich allowed separate quantitation of the peaks from sense andantisense strand.

Peak areas for full length strands were obtained by integration of theUV detector signal using software supplied by the manufacturer of theinstrument (Chromeleon 6.5; Dionex GmbH, Idstein, Germany).

6.3 Data Analysis:

Integrated sense strand, antisense strand, and internal standard peakareas were obtained for all samples and the normalization control.

A correction factor CF, accounting for liquid losses in the filtrationand washing steps, was determined for every sample by calculating theratio of experimental to theoretical internal standard peak area. Thetheoretical internal standard peak area is obtained, e.g. from acalibration curve of the internal standard obtained by injecting 20 μleach of a serial dilution of the 50 μM solution of (L-dT)₄₀ onto theHPLC column, and calculation of the theoretical peak area correspondingto 30 pmole (L-dT)₄₀ with the equation obtained by linear least squarefit to the peak areas from the dilution series. The correction factor CFto be applied to the peak areas of the sense and antisense strand isobtained as:CF=PeakArea_(intStd)(theoretical)/PeakArea_(intStd)(Sample)

This treatment assumes that, by virtue of washing the filter twice,virtually complete recovery is achieved in the combined filtrates, andcorrects for the variable volume of wash water retained in the filter,such that peak areas from different samples can be compared.

The peak areas obtained for the sense and antisense strand peaks foreach time point are then multiplied with the correction factor CF toobtain Normalized Peak Areas (NPA_(sense,t), NPA_(antisense,t)):NPA _(sense or antisense,t)=(Peak Area_(sense or antisense,t))×CF

To obtain the relative amount of remaining Full Length Product (% FLP)for the sense and antisense strands at time t, the Normalized Peak Areafor each strand at time t=0 min (NPA_(sense,t=0), NPA_(antisense,t=0))is set as 100%, and the NPAs from other time points are divided by thesevalues.%FLP _(t=1,2,3 . . . n)=(NPA _(t=1,2,3 . . . n) /NPA _(t=0))*100

The value obtained from the control sample, where the siRNA wasincubated with annealing buffer only, may serve as a control of theaccuracy of the method. The % FLP for both strands should lie near 100%,within error margins, regardless of time of incubation.

The degradation half life t_(1/2) may then be calculated for eachstrand, assuming first order kinetics, from the slope of a linear leastsquare fit to a plot of ln(% FLP) versus time as:t _(1/2)=ln(0,5)/slope7. Analysis of the Ability of Unmodified and Modified siRNAs to Inhibitthe Expression of a Target Gene7.1 Incubation of Cultured HepG2 Cells with siRNAs Specific for HumanApoB

The ability of siRNAs bearing no modifications or various combinationsof 2′-O-methyl, 2′-deoxy-2′-fluoro, and phosphorothioate linkages toinhibit the expression of human ApoB was tested. HepG2 cells in culturewere used for quantitation of ApoB mRNA in total mRNA isolated fromcells incubated with ApoB-specific siRNAs by branched DNA assay. HepG2cells were obtained from American Type Culture Collection (Rockville,Md., cat. No. HB-8065) and cultured in MEM (Gibco Invitrogen, InvitrogenGmbH, Karlsruhe, Germany, cat. No. 21090-022) supplemented to contain10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany, cat. No.S0115), 2 mM L-Glutamin (BiochromAG, Berlin, Germany, cat. No. K0238),Penicillin 100 U/ml, Streptomycin 100 μg/ml (BiochromAG, Berlin,Germany, cat. No. A2213), 1× non-essential amino acids (NEA) (BiochromAG, Berlin, Germany, cat. No. K0293) and 1 mM sodium pyruvate (BiochromAG, Berlin, Germany, cat. No. L0473) at 37° C. in an atmosphere with 5%CO₂ in a humidified incubator (Heraeus HERAcell, Kendro LaboratoryProducts, Langenselbold, Germany).

For transfection with siRNA, HepG2 cells were seeded at a density of1.5×10⁴ cells/well in 96-well plates and cultured for 24 hours.Transfection of siRNA was carried out with oligofectamine (InvitrogenGmbH, Karlsruhe, Germany, cat. No. 12252-011) as described by themanufacturer. SiRNAs were transfected at a concentration of 30 nM forthe screening of siRNA duplexes. 24 hours after transfection, the mediumwas changed and cells were incubated for an additional 24 hours. Formeasurement of ApoB mRNA by branched DNA assay, as described below,cells were harvested and lysed following procedures recommended by themanufacturer of the Quantigene Explore Kit (Genospectra, Fremont,Calif., USA, cat. No. QG-000-02) for bDNA quantitation of mRNA, exceptthat 2 μl of a 50 μg/μl stock solution of Proteinase K (Epicentre,Madison, Wis., USA, Cat. No. MPRK092) was added to 600 μl of Tissue andCell Lysis Solution (Epicentre, Madison, Wis., USA, cat. No. MTC096H).Lysates were stored at −80° C. until analysis by branched DNA assay.

AL-DP-HCV (see Table 2, SEQ ID NO. 110 and SEQ ID NO. 111) was used asnegative control.

7.2 Branched DNA Assay for the Quantitation of ApoB mRNA in CulturedHepG2 Cells

ApoB100 mRNA levels were measured by branched-DNA (bDNA) assay. Theassay was performed using the Quantigene Explore Kit (Genospectra,Fremont, Calif., USA, cat. No. QG-000-02). Frozen lysates were thawed atroom temperature, and ApoB and GAPDH mRNA quantified using theQuantigene Explore Kit according to manufacturer's instructions. Nucleicacid sequences for Capture Extender (CE), Label Extender (LE) andblocking (BL) probes were selected from the nucleic acid sequences ofApoB and GAPDH with the help of the QuantiGene ProbeDesigner Software2.0 (Genospectra, Fremont, Calif., USA, cat. No. QG-002-02). Probenucleotide sequences used in quantization of human ApoB are shown inTable 3. Probe nucleotide sequences used in quantization of human GAPDHare shown in Table 4.

The ApoB mRNA levels were normalized across different samples bycomparing the ratio of ApoB mRNA to GAPDH mRNA present in the samples.The activity of a given ApoB specific siRNA duplex was expressed as apercentage of ApoB mRNA (ApoB mRNA/GAPDH mRNA) in treated cells relativeto cells treated with the control siRNA.

TABLE 3  DNA probes for human ApoB used in branched-DNA assays SEQ.Probe ID. type^(a) Nucleotide sequence No. CE

ATTGGATTTTCAGAATACTGTATAGCTTTTTTCTCTTGGAAAGAAAGT 156 CE

CTGCTTCGTTTGCTGAGGTTTTTTCTCTTGGAAAGAAAGT 157 CE

CAGTGATGGAAGCTGCGATATTTTTCTCTTGGAAAGAAAGT 158 CE

AACTTCTAATTTGGACTCTCCTTTGTTTTTCTCTTGGAAAGAAAGT 159 CE

CTCCTTCAGAGCCAGCGGTTTTTCTCTTGGAAAGAAAGT 160 CE

CTCCCATGCTCCGTTCTCATTTTTCTCTTGGAAAGAAAGT 161 CE

GGGTAAGCTGATTGTTTATCTTGATTTTTCTCTTGGAAAGAAAGT 162 LE

GTTCCATTCCCTATGTCAGCATTTTTAGGCATAGGACCCGTGTCT 163 LE

TTAATCTTAGGGTTTGAGAGTTGTGTTTTTAGGCATAGGACCCGTGTCT 164 LE

ACTGTGTTTGATTTTCCCTCAATATTTTTAGGCATAGGACCCGTGTCT 165 LE

GTATTTTTTTCTGTGTGTAAACTTGCTTTTTAGGCATAGGACCCGTGTCT 166 LE

AATCACTCCATTACTAAGCTCCAGTTTTTAGGCATAGGACCCGTGTCT 167 BL

GCCAAAAGTAGGTACTTCAATTG 168 BL

TTGCATCTAATGTGAAAAGAGGA 169 BL

ATTTGCTTGAAAATCAAAATTGA 170 BL

GTACTTGCTGGAGAACTTCACTG 171 BL

CATTTCCAAAAAACAGCATTTC 172 ^(a)CE = Capture Extender probe; LE = LabelExtender probe; BL = blocking probe

TABLE 4 DNA probes for human GAPDH used in branched-DNA assays ProbeSEQ. ID. type^(a) Nucleotide sequence No. CE

AATTTGCCATGGGTGGAATTTTTTCTCTTGGAAAGAAAGT 173 CE

GAGGGATCTCGCTCCTGGATTTTTCTCTTGGAAAGAAAGT 174 CE

CCCAGCCTTCTCCATGGTTTTTTCTCTTGGAAAGAAAGT 175 CE

CTCCCCCCTGCAAATGAGTTTTTCTCTTGGAAAGAAAGT 176 LE

GCCTTGACGGTGCCATGTTTTTAGGCATAGGACCCGTGTCT 177 LE

ATGACAAGCTTCCCGTTCTCTTTTTAGGCATAGGACCCGTGTCT 178 LE

GATGGTGATGGGATTTCCATTTTTTTAGGCATAGGACCCGTGTCT 179 LE

CATCGCCCCACTTGATTTTTTTTTAGGCATAGGACCCGTGTCT 180 LE

ACGACGTACTCAGCGCCATTTTTAGGCATAGGACCCGTGTCT 181 LE

GCAGAGATGATGACCCTTTTGTTTTTAGGCATAGGACCCGTGTCT 182 BL

GTGAAGACGCCAGTGGACTC 183 ^(a)CE = Capture Extender probe; LE = LabelExtender probe; BL = blocking probe8. Results8.1 Results from Electrophoretic Separation of dsRNA Fragments Followedby Staining

The stability of the partially modified dsRNA of Table 2 labelledGE1s/GE1as through GE7s/GE1as was assessed using the serum incubationassay using human serum followed by analysis of fragments using the“stains all” or LC/MS procedure. A number of sequence motifs were foundto be particularly susceptible to cleavage by endonucleases present inmammalian serum. By incorporating 2′-O-methyl modified nucleotides atstrategic sites within a dsRNA it was possible to significantly increasethe stability and integrity of such molecules in biologic media such asserum.

The unmodified all-ribo dsRNA GE1s/GE1as was completely degraded within15 minutes (see FIG. 5). Even in the sample taken immediately afterbringing the dsRNA into contact with the serum, the upper bandcorresponding to the antisense strand had already almost completelydisappeared. By replacing all uridine nucleotides with 2′-O-methylmodified uridines, the GE7s/GE7 as dsRNA was stabilized such that anappreciable amount was still detectable after a 4 hour incubation inserum. Even after 24 h in serum, trace amounts of the undegraded strandswere still visible.

Introduction of two more 2′-O-methyl nucleotides at position 21 (G) and22 (C) in the single stranded 3′-overhang of the antisense strand, as incase of GE7s/GE8s, further enhanced stability, as evidenced by thestronger bands corresponding to undegraded strands at the 1, 2, and 4hour time points. Additionally introducing phosphorothioate linkagesbetween position 21 and 22 of the antisense strand GE9 as of the dsRNAGE7s/GE9 as efficiently inhibited the exonucleolytic degradation of thisstrand.

The stabilizing contribution of 2′-O-methyl could be shown to besequence dependent. By a stepwise removal of 2′-O-methyl uridines thecrucial sequence context could be identified as sequences of 5′-ua-3′.In GE7s/GE10as, position 8 in the anti-sense strand contains a regularuridine residue. After 30 minutes in the serum incubation assay adegraded species could be detected which resulted from anendonucleolytic degradation process between positions 7 and 8 of theantisense strand. In the GE7s/GE11as dsRNA, this position is substitutedby a 2′-O-methyl uridine and, consequently, the formation of theaforementioned degradation product could not be observed. Furthermore,two positions in GE7s/GE11 as, namely positions 6 and 20, demonstratedthat uridines in a 5′-AU-3′ sequence context need not necessarily beexchanged by 2′-O-methyl uridines in order to enhance resistance towardsdegradation by nucleases. Duplexes of unmodified GE1s with the modifiedantisense strands GE7 as, GE8 as; GE9 as; GE10as and GE11 as,respectively, were rapidly degraded demonstrating the requirement thatboth strands need to be modified for an enhanced stability (see FIGS.10-14). The rapid degradation of GE7s/GE1as revealed that the samerequirements are valid for the protection of the antisense strand fromdegradation.

8.2 Results of Separation and Identification of dsRNA Fragments by LC/MS

Analysis of the serum degradation fragments of duplex LC1s/LC1as afterincubation with mouse serum using LC/MS yielded the fragments listed inTable 5 and Table 6.

TABLE 5 Fragments identified by LC/MS after mouse serum incubation of LC1s/LC1as derived from the antisense strand 5′-

cgaaguacucagcguaagTT-3′ (SEQ ID NO: 11); predicted mass [M − H] =6692.1 Da   2′/3′ terminal pred. obs. SEQ ID Position Sequence phosphateMass Mass NO: U1-T20

cgaaguacucagcguaagT No 6387.9 6386.0 112 U1-G19

cgaaguacucagcguaag No 6083.8 6080.7 113 U1-U16

cgaaguacucagcgu No 5160.1 5160.6 114 U1-U16

cgaaguacucagcgu-tp cyclic 5142.1 5142.1 115 U1-U7

cgaagu-tp cyclic 2265.4 2265.5 116 A17-T21

agTT No 1549.0 1549.1 117

TABLE 6 Fragments identified by LC/MS after mouse serumincubation of LC1s/LC1as derived from the sense strand 5′-

uuacgcugaguacuucgaTT-3′ (SEQ ID NO: 10); predicted mass [M − H] =6606.0 Da   2′/3′ terminal pred. obs. SEQ ID Position Sequence phosphateMass Mass NO C1-T20

uuacgcugaguacuucgaT no 6301.8 6301.1 118 C1-A19

uuacgcugaguacuucga no 6083.8 6080.7 119 A4-T21

cgcugaguacuucgaTT no 5688.5 5688.8 120 A4-T20

cgcugaguacuucgaT no 5384.4 5385.9 121 A4-U12

cgcugagu-tp cyclic 2915.8 2916.1 122

Thus, the main sites of endonucleolytic cleavage identified by LC/MS inLC1s/LC1as were the dinucleotides 5′-ua-3′ in positions 7-8 and 16-17 ofthe antisense strand, and positions 4-5 and 12-13 in the sense strand,counting from the 5′-end of the respective strand. In addition, stepwiseexonucleolytic cleavage of the 3′-TT-overhangs was detected in bothstrands.

Analysis of the serum degradation fragments of duplex LC1s/LC1as usingLC/MS yielded the fragments listed in Table 7 and Table 8.

TABLE 7 Fragments identified by LC/MS after mouse serumincubation of LC2s/LC2as derived from the antisensestrand uuggugagguuugauccgcTT (SEQ ID NO: 13); calculated Mass: [M − H] =6679.0 Da 2′/3′ terminal pred. obs. SEQ ID Position Sequence phosphateMass Mass NO: u2-T21

uggugagguuugauccgcTT no 6376.8 6373.8 123 u1-T20

uggugagguuugauccgcT no 6374.8 6373.8 124 u1-c19

uggugagguuugauccgc no 6070.6 6070.5 125 u2-T20

uggugagguuugauccgcTT no 6068.7 6070.5 126 g3-T21

gugagguuugauccgcTT no 6066.7 6067 127 g3-T20

gugagguuugauccgcT no 5762.5 5763 128 u5-T20

gagguuugauccgcT no 5072.1 5071 129 g6-T21

agguuugauccgcTT no 5070.1 5071 130 u1-u11

uggugagguu-tp 2′-/3′- 3603.1 3605 131 phosphate u1-u11

uggugagguu-tp cyclic 3585.1 3586.8 132 u1-u10

uggugaggu-tp 2′-/3′- 3297 3298.5 133 phosphate u1-u10

uggugaggu-tp cyclic 3279 3280 134 u12-T21

gauccgcTT no 3093 3094 135 g3-u11

gugagguu-tp 2′-/3′- 2990.8 2993 136 phosphate g3-u11

gugagguu-tp cyclic 2972.8 2974.3 137 u12-T20

gauccgcT no 2788.7 2788 138 g13-T21

auccgcTT no 2786.8 2788 139 g6-u12

agguuu-tp cyclic 2282.3 2282 140 u5-u11

gagguu-tp cyclic 2282.3 2282 141

TABLE 8 Fragments identified by LC/MS after mouse serumincubation of LC2s/LC2as derived from the sensestrand gcggaucaaaccucaccaaTT (SEQ ID NO: 12); calculated Mass: [M − H] =6635.1 Da 2′/3′ terminal cal. exp. SEQ ID Position Sequence phosphateMass Mass NO: g1-T20

cggaucaaaccucaccaaT no 6329.9 6330.5 142 g1-c17

cggaucaaaccucacc cyclic 5429.3 5429.4 143 g1-c16

cggaucaaaccucac cyclic 5124.1 5124.8 144 g1-c14

cggaucaaaccuc cyclic 4489.8 4490 145 c7-T20

aaaccucaccaaT no 4353.8 4354 146 a8-T21

aaccucaccaaTT no 4352.8 4354 147 c7-a19

aaaccucaccaa no 4049.5 4050 148 a8-T20

aaccucaccaaT no 4048.5 4050 149 a8-c17

aaccucacc cyclic 3147.9 3149 150 c7-c16

aaaccucac cyclic 3147.9 3149 151 a8-c16

aaccucac cyclic 2842.8 2843.8 152 g1-c7

cggauc cyclic 2280.4 2282 153 a8-c14

aaccuc cyclic 2208.4 2209.3 154 g1-u6

cggau cyclic 1975.2 1977 155

Table 7 shows that LC2s/LC2 as was subject to some exonucleolyticdegradation. The fragments corresponding to u2-T21/ul-T20 andu2-T20/u1-c19 cannot be unequivocally assigned due to their small massdifference of 2 Da, but the observed mass is closer to the ul-T20 andu1-c19. Hence, it seems likely that exonucleases present in mouse serumare able to degrade the terminal TT single strand overhang.Exonucleolytic activity in mouse serum was generally greater than inhuman serum.

LC2s/LC2as was chosen for analysis partly because its sense strand doesnot comprise the 5′-ua-3′ motif, and therefore could potentially yieldinformation on other sequence motifs prone to endonucleolytic attack.Indeed, the fragments g3-T21, g3-T20, g3-ul1, g6-T21, g6-u12, andg13-T21 point towards 5′-ug-3′ as a point of efficient degradativeaction by endonucleases. Similarly, the fragments u1-u10, u1-u11, bothinvolving a terminal phosphate, and u12-u21 indicate 5′-uu-3′ as a thirdmotif of endonucleolysis. In a different experiment (data not shown),5′-ca-3′,5′-cc-3′,5′-cu-3′ and 5′-uc-3′ were further shown occasionallyto be targets of endonucleolytic degradation.

Accordingly, a number of siRNA duplexes were synthesized, wherein thepyrimidine nucleotides present in a sequence context of5′-ua-3′,5′-ug-3′ and 5′-uu-3′ were protected towards endonucleolyticdegradation by 2′-O-methyl modifications. In addition, for incubationwith mouse serum, the 3′-terminus of the sense strand was protectedagainst exonucleolytic degradation by conjugation to a cholesterylmoiety via a pyrrolidinyl phosphorothiodiester. AL-DP 5542 is identicalin sequence to AL-DP 5543 but for these potentially stabilizing2′-modifications in AL-DP 5542. AL-DP 5544 is identical in sequence toAL-DP 5545 but for the same potentially stabilizing 2′-modifications inAL-DP 5544.

Degradation half lives were determined for each of the RNA strands ofAL-DP 5542, AL-DP 5543, AL-DP 5544, and AL-DP 5545 as described undersection 6 above. Results are given Table 9.

TABLE 9 Half life in hours for the degradation of individual strands ofAL-DP 5542, AL-DP 5543, AL-DP 5544, and AL-DP 5545 measured by HPLCafter incubation of the RNA duplex with mouse serum Half life in mouseserum (hours) AL-DP 5542 AL-DP 5543 AL-DP 5544 AL-DP 5545 sense strand36 1.6 21 3.6 antisense strand 8 0.5 19 8

Table 9 shows, that the 2′-modification of nucleotides in sequencecontexts making them prone to degradation led to a stabilization of theindividual strands by a factor between about 2 and about 20.

By replacement of all 5′-u and 5′-c in sequence motifs5′-ua-3′,5′-ug-3′,5′-ca-3′,5′-uu-3′,5′-uc-3′,5′-cc-3′, and 5′-cu-3′siRNA duplexes with very high stability towards endonucleolyticdegradation could be generated. By stepwise replacement of first all5′-U and 5′-C in sequence motifs 5′-ua-3′, then, for example, all 5′-uin sequence motifs 5′-ug-3′, then, for example, all 5′-c in sequencemotifs 5′-ca-3′, then, for example, all 5′-u in sequence motifs5′-uu-3′, then, for example, all 5′-u in sequence motifs 5′-uc-3′, then,for example, all 5′-u in sequence motifs 5′-cc-3′, and lastly, forexample, all 5′-c in sequence motifs 5′-cu-3′, intermediately assessingstability towards nucleolytic degradation in serum, it was possible totailor-make dsRNA duplexes with a certain desired stability in serum.Where it was necessary to keep the number of modifications to a minimumin order to preserve the activity of a certain siRNA for inhibition oftarget gene expression while attaining a certain minimum stability, thisstepwise approach was successfully applied by replacing only the mostvulnerable sites with modified nucleotides until the best compromisebetween stability and activity was obtained.

8.3 Stability and Activity of siRNAs Bearing 2′-Modifications in5′-Uridines and 5′-Cytidines in Sequence Motifs5′-ua-3′,5′-ug-3′,5′-ca-3′, and 5′-uu-3′

Good stability with reasonable or no loss of gene expression inhibitingactivity compared to the all-ribonucleotide sequence was usuallyobtained when modifying 5′-uridines and 5′-cytidines in sequence motifs5′-ua-3′,5′-ug-3′,5′-ca-3′, and 5′-uu-3′.

FIG. 23 to FIG. 36 show comparisons of the stability of siRNAsAL-DP-5097, AL-DP-5398, AL-DP-5458, AL-DP-5098, AL-DP-5399, AL-DP-5459,AL-DP-5024, AL-DP-5388, AL-DP-5448, AL-DP-5013, AL-DP-5387, AL-DP-5447,AL-DP-5084, AL-DP-5394, AL-DP-5454, AL-DP-5094, AL-DP-5397, AL-DP-5457,AL-DP-5093, AL-DP-5396, AL-DP-5456, AL-DP-5089, AL-DP-5395, AL-DP-5455,AL-DP-5030, AL-DP-5389, AL-DP-5449, AL-DP-5035, AL-DP-5390, AL-DP-5450,AL-DP-5046, AL-DP-5391, AL-DP-5451, AL-DP-5048, AL-DP-5392, AL-DP-5452,AL-DP-5002, AL-DP-5386, AL-DP-5446, AL-DP-5049, AL-DP-5393, andAL-DP-5453, after incubation with human serum and analysis byelectrophoresis and “stains all” detection. Table 10 lists thecorresponding activities of these siRNAs towards the reduction of ApoBmRNA concentrations present in cultured HepG2 cells as compared to cellsincubated with unrelated siRNA AL-DP-HCV.

FIG. 23 to FIG. 36 show that the unmodified siRNAs AL-DP-5097,AL-DP-5098, AL-DP-5024, AL-DP-5013, AL-DP-5084, AL-DP-5094, AL-DP-5093,AL-DP-5089, AL-DP-5030, AL-DP-5035, AL-DP-5046, AL-DP-5048, AL-DP-5002,and AL-DP-5049 are degraded very quickly, such that after 1 hour ofserum incubation, virtually no full length strands are detectable. Bycontrast, the corresponding modified siRNAs show a marked increase instability under the conditions of serum incubation, full length senseand antisense strands remaining almost undegraded for at least 6 hoursof incubation.

TABLE 10 Activities of siRNAs bearing various modifications towards thereduction of ApoB mRNA concentrations present in cultured HepG2 cells ascompared to cells incubated with unrelated siRNA AL-DP-HCV Start codonof ApoB mRNA sequence in ApoB remaining in cells mRNA incubated withDuplex complementary to siRNA in % of Modification Descriptor antisensestrand control^(a) pattern^(b) AL-DP-5024  465 13 Unmodified AL-DP-5388″ 17 2′-O-methyl AL-DP-5448 ″ 20 2′-deoxy-2′-fluoro AL-DP-5013  466 17Unmodified AL-DP-5387 ″ 40 2′-O-methyl AL-DP-5447 ″ 242′-deoxy-2′-fluoro AL-DP-5084  623 36 Unmodified AL-DP-5394 ″ 362′-O-methyl AL-DP-5454 ″ 68 2′-deoxy-2′-fluoro AL-DP-5094 1287 23Unmodified AL-DP-5397 ″ 42 2′-O-methyl AL-DP-5457 ″ n.d.2′-deoxy-2′-fluoro AL-DP-5097 1296 15 Unmodified AL-DP-5398 ″ 212′-O-methyl AL-DP-5458 ″ 19 2′-deoxy-2′-fluoro AL-DP-5093 1339 26Unmodified AL-DP-5396 ″ 93 2′-O-methyl AL-DP-5456 ″ 352′-deoxy-2′-fluoro AL-DP-5098 2133 21 Unmodified AL-DP-5399 ″ 202′-O-methyl AL-DP-5459 ″ 26 2′-deoxy-2′-fluoro AL-DP-5089 2807 32Unmodified AL-DP-5395 ″ 25 2′-O-methyl AL-DP-5455 ″ 342′-deoxy-2′-fluoro AL-DP-5030 2866 12 Unmodified AL-DP-5389 ″ 412′-O-methyl AL-DP-5449 ″ 18 2′-deoxy-2′-fluoro AL-DP-5035 2869 17Unmodified AL-DP-5390 ″ 21 2′-O-methyl AL-DP-5450 ″ 222′-deoxy-2′-fluoro AL-DP-5046 10180  19 Unmodified AL-DP-5391 ″ 452′-O-methyl AL-DP-5451 ″ 42 2′-deoxy-2′-fluoro AL-DP-5048 10187  19Unmodified AL-DP-5392 ″ 45 2′-O-methyl AL-DP-5452 ″ 262′-deoxy-2′-fluoro AL-DP-5002 13539  19 Unmodified AL-DP-5386 ″ 542′-O-methyl AL-DP-5446 ″ 21 2′-deoxy-2′-fluoro AL-DP-5049 13550  24Unmodified AL-DP-5393 ″ 69 2′-O-methyl AL-DP-5453 ″ 412′-deoxy-2′-fluoro ^(a)n.d. not determined ^(b)2′-O-methyl =2′-O-methyl-modifications of the 5′-uridine or 5′-cytidine in alloccurrences of the sequence motifs 5′-ua-3′, 5′-ug-3′, 5′-ca-3′, and5′-uu-3′, as well as in positions 22 and 23 of the antisense strand,phosphorothioate linkages between positions 20 an 21 of the sensestrand, and between positions 22 and 23 of the antisense strand;2′-deoxy-2′-fluoro: 2′-deoxy-2′-fluoro-modifications of the 5′-uridineor 5′-cytidine in all occurrences of the sequence motifs 5′-ua-3′,5′-ug-3′, 5′-ca-3′, and 5′-uu-3′, 2′-O-methyl-modifications in positions22 and 23 of the antisense strand unless already2′-deoxy-2′-fluoro-modified, phosphorothioate linkages between positions20 an 21 of the sense strand, and between positions 22 and 23 of theantisense strand

At the same time, Table 10 shows that the gene silencing activity of theso modified siRNAs is only slightly to moderately reduced, where the2′-O-methyl modification may have a slightly greater impact than the2′-deoxy-2′-fluoro-modification.

1. A double-stranded ribonucleic acid (dsRNA) having a sense strand andan antisense strand and having increased stability in a biologicalsample, wherein the sense strand and the antisense strand are bothmodified, said antisense-strand modifications consisting of: (a)2′-modified uridines in all occurrences of the sequence motif5′-uridine-adenine-3′ (5′-ua-3′), and (b) 2′-modified cytidines in alloccurrences of the sequence motif 5′-cytidine-adenine-3′ (5′-ca-3′). 2.The dsRNA of claim 1, wherein the sense-strand modifications comprise atleast one of the features (a) and (b).
 3. The dsRNA of claim 1, whereinthe dsRNA comprises at least two occurrences of at least one of thesequence motifs.
 4. The dsRNA of claim 1, wherein the dsRNA comprises atleast three occurrences of at least one of the sequence motifs.
 5. ThedsRNA of claim 1, wherein the sense strand further comprises at leastone dinucleotide selected from the group consisting of 5′-uu-3′,5′-ug-3′, 5′-uc-3′, 5′-cu-3′, 5′-cg-3′, and 5′-cc-3′, wherein nonucleotide of the at least one dinucletide is a 2′-O-modifiednucleotide.
 6. The dsRNA of claim 1, wherein the 2′-modified nucleotideis a 2′-deoxy nucleotide, a 2′-O-methyl nucleotide, a 2′-deoxyfluoronucleotide, a 2′-O-methoxyethyl (2′-O-MOE) nucleotide, a2′-O—N-methylacetamido (2′-O-NMA) nucleotide, a2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, a2′-O-aminopropyl (2′-O-AP) nucleotide, a 2′-ara-F nucleotide, or alocked nucleic acid nucleotide.
 7. The dsRNA of claim 2, wherein thesense strand mocifications further comprising at least one of (c)2′-modified uridines in all occurrences of the sequence motif5′-uridine-guanine-3′ (5′-ug-3′), or (d) 2′-modified 5′-most uridines inall occurrences of the sequence motif 5′-uridine-uridine-3′ (5′-uu-3′),or (e) 2′-modified 5′-most cytidines in all occurrences of the sequencemotif 5′-cytidine-cytidine-3′ (5′-cc-3′), or (f) 2′-modified cytidinesin all occurrences of the sequence motif 5′-cytidine-uridine-3′(5′-cu-3′), or (g) 2′-modified uridines in all occurrences of thesequence motif 5′-uridine-cytidine-3′ (5′-uc-3′).
 8. The dsRNA of claim7, wherein the sense strand comprises at least one of features (c) and(d).
 9. The dsRNA of claim 7, wherein the sense strand comprisesfeatures (c) and (d).
 10. A method of preparing a pharmaceuticalcomposition, comprising formulating the dsRNA of claim 1 in apharmaceutically acceptable carrier.
 11. A pharmaceutical compositionfor inhibiting the expression of a target gene in a mammal, comprising:(a) at least one dsRNA of claim 1; and (b) a pharmaceutically acceptablecarrier.
 12. A double-stranded ribonucleic acid (dsRNA) having a sensestrand and an antisense strand and having increased stability in abiological sample, wherein the sense strand and the antisense strand areboth 2′-modified, said antisense-strand 2′-modifications consisting of:(a) 2′-modified uridines in all occurrences of the sequence motif5′-uridine-adenine-3′ (5′-ua-3′), and (b) 2′-modified cytidines in alloccurrences of the sequence motif 5′-cytidine-adenine-3′ (5′-ca-3′). 13.The dsRNA of claim 12, wherein the sense-strand modifications compriseat least one of the features (a) and (b).
 14. The dsRNA of claim 12,wherein the dsRNA comprises at least two occurrences of at least one ofthe sequence motifs.
 15. The dsRNA of claim 12, wherein the dsRNAcomprises at least three occurrences of at least one of the sequencemotifs.
 16. The dsRNA of claim 12, wherein the sense strand furthercomprises at least one dinucleotide selected from the group consistingof 5′-uu-3′, 5′-ug-3′, 5′-uc-3′, 5′-cu-3′, 5′-cg-3′, and 5′-cc-3′,wherein no nucleotide of the at least one dinucleotide is a2′-O-modified nucleotide.
 17. The dsRNA of claim 12, wherein the2′-modified nucleotide is a 2′-deoxy nucleotide, a 2′-O-methylnucleotide, a 2′-deoxyfluoro nucleotide, a 2′-O-methoxyethyl (2′-O-MOE)nucleotide, a 2′-O-N-methylacetamido (2′-O-NMA) nucleotide, a2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, a2′-O-aminopropyl (2′-O-AP) nucleotide, a 2′-ara-F nucleotide, or alocked nucleic acid nucleotide.
 18. The dsRNA of claim 13, wherein thesense strand 2′-modifications further comprise at least one of (c)2′-modified uridines in all occurrences of the sequence motif5′-uridine-guanine-3′ (5′-ug-3′), or (d) 2′-modified 5′-most uridines inall occurrences of the sequence motif 5′-uridine-uridine-3′ (5′-uu-3′),or (e) 2′-modified 5′-most cytidines in all occurrences of the sequencemotif 5′-cytidine-cytidine-3′ (5′-cc-3′), or (f) 2′-modified cytidinesin all occurrences of the sequence motif 5′-cytidine-uridine-3′(5′-cu-3′), or (g) 2′-modified uridines in all occurrences of thesequence motif 5′-uridine-cytidine-3′ (5′-uc-3′).
 19. The dsRNA of claim7, wherein the sense strand comprises at least one of features (c) and(d).
 20. The dsRNA of claim 7, wherein the sense strand comprisesfeatures (c) and (d).
 21. A method of preparing a pharmaceuticalcomposition, comprising formulating the dsRNA of claim 12 in apharmaceutically acceptable carrier.
 22. A pharmaceutical compositionfor inhibiting the expression of a target gene in a mammal, comprising:(a) at least one dsRNA of claim 12; and (b) a pharmaceuticallyacceptable carrier.